Novel biologically active peptides and their new uses

ABSTRACT

Novel peptides are disclosed with their use as a pharmaceutical composition. A method is also disclosed for making pharmaceutical compositions and treatment of an individual.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to short peptides and the use thereof.In particular, the present invention is related to short peptides withbiological activities.

2. Description of the Related Art

Peptides are known in the art for treatment of diseases and aspharmaceutical compositions. For example, U.S. Pat. No. 6,191,113discloses a peptide that has inhibitory activity for the growth ofsmooth muscle cells and is therefore useful for preventing and treatingpathological conditions associated with growth of smooth muscle cellssuch as arteriosclerosis, restenosis after angioplasty, luminal stenosisafter grafting blood vessel and smooth muscle sarcoma. U.S. Pat. No.6,184,208 discloses another peptide that is found to modulatephysiological processes such as weight gain activity of the epithelialgrowth zone and hair growth. Furthermore, PCT publication No. WO03/006492 and U.S. patent application Ser. No. 10/237,405 suggested thatcertain peptides and their pharmaceutical compositions are biologicallyactive and capable of modulating immune responses.

It is therefore an object of the present invention to provide a shortpeptide or peptides that have biological activity.

SUMMARY OF THE INVENTION

The peptides according to the present invention were individuallysynthesized by standard chemical methods. Using known animal and invitro methods, various biological functions of these peptides wereanalyzed using methods described in the following references asdescribed below. The peptide names are given the code “CMS” followed bya number. The peptide sequence and the corresponding ID numbers areshown in Table 1. A total of 7 new peptides have been identified ashaving in vivo biological activities, and indicated with an asterisknext to the sequence ID Number. The remaining peptides have beenreported previously in WIPO publication WO 03/006492, but novel andinventive new indications have been found in the present invention. Forease of reference, the sequences that are found in WO 03/006492 havebeen given the same sequence ID number.

TABLE 1 Sequence Listing Peptide ID No. Name Peptide Sequence  1 CMS-001Pro Thr Thr Lys Thr Tyr Phe Pro His Phe  2 CMS-017Ile-Val-Thr-Asn-Thr-Thr  3 CMS-008 Lys Ala Val Gly His Leu Asp Asp LeuPro Gly Ala Leu  4* CMS001.30 Pro-Thr-Thr-Lys-Thr-Tyr  5* CMS001.31Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro  6 No sequence  7 CMS-014 Ala Ala HisHis Pro Asp Asp Phe Asn Pro Ser Val  8* CMS024.04 Tyr-Ser-Nle where Nle= norleucine (2-aminohexanoic acid)  9* CMS024.05 Tyr-Thr-Val  10*CMS024.14 (3,5-dibromo-Tyr)-Ser-Leu  11* CMS024.16 Leu-Tyr-Ser 12 Nosequence 13 No sequence 14 No sequence 15 CMS-023 Ala Ala Phe 16 Nosequence 17 No sequence 18 No sequence 19 No sequence 20 No sequence 21CMS-030 Phe Glu Glu Met

Accordingly, one aspect of the present invention relates tosubstantially pure peptides having sequences identified as SEQ ID Nos.4, 5, 8-11. Thus the present invention also relates to a substantiallypure peptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID Nos. 4, 5, 8-11. It also relates to a substantiallypure peptide consisting essentially of an amino acid sequence selectedfrom the group consisting of SEQ ID Nos. 4, 5, 8-11. The invention alsorelates to a substantially pure peptide consisting of an amino acidsequence selected from the group consisting of SEQ ID Nos. 4, 5, 8-11.In a specific embodiment, the peptides can modulate, but are not limitedto modulating, one or more of the following: fatigue, nutritionaldisorders, metabolic disorders, lipid metabolism disorder, acidosis,immune activity, effects of radiation, hepatitis, rejection oftransplanted organ, the growth of cancer and appetite.

In the more preferred embodiment, the peptides can modulate, but are notlimited to modulating one or more of the following conditions: liverglycogen storage, blood lactic acid levels, radiotherapy-inducedimmunodeficiency, hepatitis B infection, skin graft rejection, livercancer, stomach cancer, inflammation, blood lipid, triglyceride andtotal cholesterol, immune hypersensitivity, T-lymphocyte proliferation,mixed lymphocyte proliferation, obesity, and higher than normal bodyweight.

Another aspect of the present invention relates to substantially purepeptides that are functional derivatives of peptides having sequencesidentified as sequence ID No. 4, 5, 8-11. Thus the present inventionrelates also to a substantially pure peptide comprising an amino acidsequence which is a functional derivative of a biologically activepeptide, this biologically active peptide having an amino acid sequenceselected from the group consisting of SEQ ID Nos. 4, 5, 8-11. It alsorelates to a substantially pure peptide consisting essentially of anamino acid sequence which is a functional derivative of a biologicallyactive peptide, this biologically active peptide having an amino acidsequence selected from the group consisting of SEQ ID Nos. 4, 5, 8-11.The invention also relates to a substantially pure peptide consisting ofan amino acid sequence which is a functional derivative of abiologically active peptide, this biologically active peptide having anamino acid sequence selected from the group consisting of SEQ ID Nos. 4,5, 8-11. In a specific embodiment, the peptides that are functionalderivatives can modulate, but not limited to modulating, one or more ofthe following: fatigue, nutritional disorders, metabolic disorders,lipid metabolism disorder, acidosis, immune activity, effects ofradiation, hepatitis, rejection of transplanted organ, the growth ofcancer and appetite.

Another aspect of the present invention relates to a genetic vectorcomprising a nucleotide sequence encoding a peptide comprising an aminoacid sequence selected from the group consisting of a peptide comprisingone of SEQ ID Nos. 4, 5, 8-11. It also relates to a genetic vectorcomprising a nucleotide sequence encoding a peptide consistingessentially of an amino acid sequence selected from the group consistingof a peptide comprising SEQ ID Nos. 4, 5, 8-11. The invention alsorelates to a genetic vector comprising a nucleotide sequence encoding apeptide comprising a functional derivative of a biologically activeamino acid sequence selected from the group consisting of SEQ ID Nos. 4,5, 8-11. It also relates to a genetic vector comprising a nucleotidesequence encoding a peptide consisting essentially of an amino acidsequence which is a functional derivative of a biologically active aminoacid sequence selected from the group consisting of SEQ ID Nos. 4, 5,8-11.

Yet another aspect of the present invention relates to hybrid peptidescontaining a leader peptide adjacent a peptide, the peptide comprising,consisting essentially of, or consisting of an amino acid sequenceselected from the group consisting of SEQ ID Nos. 4, 5, 8-11. Thepresent invention also relates to hybrid peptides containing a leaderpeptide adjacent a peptide, the peptide comprising a functionalderivative of a biologically active peptide, this biologically activepeptide comprising, consisting essentially of, or consisting of an aminoacid sequence selected from the group consisting of SEQ ID Nos. 4, 5,8-11.

The present invention also relates to a genetic vector comprising anucleotide sequence encoding a peptide comprising a leader amino acidsequence adjacent to a peptide comprising an amino acid sequence whichis a functional derivative of a biologically active amino acid sequenceselected from the group consisting of SEQ ID Nos. 4, 5, 8-11. It alsorelates to a genetic vector comprising a nucleotide sequence encoding apeptide comprising a leader amino acid sequence adjacent to a nucleotidesequence encoding a peptide consisting essentially of an amino acidsequence which is a functional derivative of a biologically active aminoacid sequence selected from the group consisting of SEQ ID Nos. 4, 5,8-11.

In a specific embodiment, the peptides produced in any of theabove-described genetic vectors can modulate, but are not limited tomodulating, one or more of the following: fatigue, nutritionaldisorders, metabolic disorders, lipid metabolism disorder, acidosis,immune activity, effects of radiation, hepatitis, rejection oftransplanted organ, the growth of cancer and appetite.

Yet another aspect of the present invention relates to a micro-organismwith a genome comprising a nucleotide sequence encoding a peptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID Nos. 4, 5, 8-11. It also relates to a micro-organism with agenome comprising a nucleotide sequence encoding a peptide consistingessentially of an amino acid sequence selected from the group consistingof SEQ ID Nos. 4, 5, 8-11.

Yet another aspect of the present invention relates to a micro-organismwith genetic material comprising a nucleotide sequence encoding apeptide, preferably an exogenous peptide comprising an amino acidsequence which is a functional derivative of a biologically active aminoacid sequence selected from the group consisting of SEQ ID Nos. 4, 5,8-11. It also relates to a micro-organism with a genetic compositioncomprising a nucleotide sequence encoding an exogenous peptideconsisting essentially of an amino acid sequence which is a functionalderivative of a biologically active amino acid sequence selected fromthe group consisting of SEQ ID Nos. 4, 5, 8-11. Exogenous peptide asused herein refers to a peptide having an amino acid sequence that isdifferent from any other peptides normally expressed by themicro-organism in its natural, unmodified form.

Yet another aspect of the present invention relates to a micro-organismwith a genetic composition comprising a nucleotide sequence encoding apeptide, preferably an exogenous hybrid peptide comprising a leaderamino acid sequence adjacent a peptide, the peptide comprising an aminoacid sequence selected from the group consisting of SEQ ID Nos. 4, 5,8-11. It also relates to a micro-organism with a genome comprising anucleotide sequence encoding a hybrid peptide comprising a leader aminoacid sequence adjacent to a peptide consisting essentially of orconsisting of an amino acid sequence selected from the group consistingof SEQ ID Nos. 4, 5, 8-11.

Yet another aspect of the present invention relates to a micro-organismwith a genetic composition comprising a nucleotide sequence encoding anexogenous hybrid peptide comprising a leader amino acid sequenceadjacent to a peptide, the peptide comprising an amino acid sequencewhich is a functional derivative of a biologically active amino acidsequence selected from the group consisting of SEQ ID Nos. 4, 5, 8-11.It also relates to a micro-organism with a genetic compositioncomprising a nucleotide sequence encoding an exogenous hybrid peptidecomprising a leader amino acid sequence adjacent a peptide consistingessentially of or consisting of an amino acid sequence which is afunctional derivative of a biologically active amino acid sequenceselected from the group consisting of SEQ ID Nos. 4, 5, 8-11.

In a specific embodiment, the peptides produced in any of theabove-described micro-organism can modulate, but are not limited tomodulating, one or more of the following: fatigue, nutritionaldisorders, metabolic disorders, lipid metabolism disorder, acidosis,immune activity, effects of radiation, hepatitis, rejection oftransplanted organ, the growth of cancer and appetite.

Yet another aspect of the present invention relates to a pharmaceuticalcomposition comprising a substantially pure peptide comprising an aminoacid sequence selected from the group consisting of SEQ ID Nos. 4, 5,8-11. The invention also relates to pharmaceutical compositioncomprising a substantially pure peptide consisting essentially of orconsisting of an amino acid sequence selected from the group consistingof SEQ ID Nos. 4, 5, 8-11.

The present invention also relates to a pharmaceutical compositioncomprising a substantially pure peptide comprising a functionalderivative of a biologically active peptide, this biologically activepeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID Nos. 4, 5, 8-11. It also relates to apharmaceutical composition comprising a substantially pure peptideconsisting essentially of a functional derivative of a biologicallyactive peptide, this biologically active peptide having an amino acidsequence selected from the group consisting of SEQ ID Nos. 4, 5, 8-11.It further relates to pharmaceutical composition comprising asubstantially pure peptide consisting of functional derivative of abiologically active peptide, this biologically active peptide having anamino acid sequence selected from the group consisting of SEQ ID Nos. 4,5, 8-11.

In a specific embodiment, the peptides present in any of theabove-described pharmaceutical compositions can modulate, but notlimited to modulating, one or more of the following: fatigue,nutritional disorders, metabolic disorders, lipid metabolism disorder,acidosis, immune activity, effects of radiation, hepatitis, rejection oftransplanted organ, the growth of cancer and appetite.

Yet a further aspect of the present invention relates to a method ofmaking a pharmaceutical composition comprising providing a substantiallypure peptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID Nos. 4, 5, 8-11; and admixing said substantiallypure peptide with a pharmaceutically acceptable carrier. It also relatesto a method of making a pharmaceutical composition comprising providinga substantially pure peptide consisting essentially of or consisting ofan amino acid sequence selected from the group consisting of SEQ ID Nos.4, 5, 8-11 and admixing said substantially pure peptide with apharmaceutically acceptable carrier.

Another aspect of the present invention is a method of making apharmaceutical composition comprising providing a substantially purepeptide comprising an amino acid sequence which is a functionalderivative of a biologically active peptide, this biologically activepeptide having an amino acid sequence selected from the group consistingof SEQ ID Nos. 4, 5, 8-11; and mixing said substantially pure peptidewith a pharmaceutically acceptable carrier.

It further relates to a method of making a pharmaceutical compositioncomprising providing a substantially pure peptide consisting essentiallyof or consisting of an amino acid sequence which is a functionalderivative of a biologically active peptide, this biologically activepeptide having an amino acid sequence selected from the group consistingof SEQ ID Nos. 4, 5, 8-11; and mixing the substantially pure peptidewith a pharmaceutically acceptable carrier.

In connection with any of the above-described method, the peptide canmodulate, but is not limited to modulating, one or more of thefollowing: immune activity, fatigue, nutritional disorders, metabolicdisorders, lipid metabolism disorder, acidosis, immune activity, effectsof radiation, hepatitis, rejection of transplanted organ, the growth ofcancer and appetite.

Yet a further aspect of the present invention relates to a method oftreatment of a human comprising administering a pharmaceuticallyeffective dose of a substantially pure peptide comprising, consistingessentially of, or consisting of an amino acid sequence selected fromthe group consisting of SEQ ID Nos. 4, 5, 8-11 to a human. It alsorelates to a method of treatment of a human comprising administering apharmaceutically effective dose of a substantially pure peptidecomprising, consisting essentially of, or consisting of an amino acidsequence which is a functional derivative of a biologically activepeptide, this biologically active peptide having an amino acid sequenceselected from the group consisting of SEQ ID Nos. 4, 5, 8-11.

In a specific embodiment, the peptides used for the treatment of humandescribed above may be used to modulate, but are not limited tomodulating, one or more of the following human conditions: fatigue,nutritional disorders, metabolic disorders, lipid metabolism disorder,acidosis, immune activity, effects of radiation, hepatitis, rejection oftransplanted organ, the growth of cancer and appetite.

In connection with any of the above-described nucleic acid sequences,the peptides and/or hybrid peptides expressed from these nucleic acidsequences can modulate, but are not limited to modulating, thefollowing: fatigue, nutritional disorders, metabolic disorders, lipidmetabolism disorder, acidosis, immune activity, effects of radiation,hepatitis, rejection of transplanted organ, the growth of cancer andappetite.

In yet another aspect, the present invention relates to the use of abiologically active peptide, said biologically active peptide having anamino acid sequence comprising, consisting essentially of, or consistingof an amino acid sequence selected from the group consisting of SEQ IDNo. 1, 2, 15 or functional derivatives thereof in the manufacture of amedicament for modulating at least one of the following conditions:hepatitis, fatigue, liver glycogen storage level, blood lactic acidlevel and immune reaction of an individual. It also relates to a methodof modulating hepatitis, liver glycogen storage level, blood lactic acidlevel or immune reaction of an individual comprising administering apharmaceutically effective dose of a biologically active peptide, saidbiologically active peptide having an amino acid sequence consistingessentially of an amino acid sequence selected from the group consistingof SEQ ID No. 1, 2, 15 or functional derivatives thereof.

The present invention further relates to the use of a biologicallyactive peptide, said biologically active peptide having an amino acidsequence comprising, consisting essentially of, or consisting of anamino acid selected from the group consisting of SEQ ID No. 3, 7 or 21or functional derivatives thereof in the manufacture of a medicament formodulating at least one of the following conditions: fatigueinflammation, appetite, body weight, body fat, blood lipid level, bloodtriglyceride level, blood cholesterol level, inflammation and immunity.The present invention also relates to a method of modulating at leastone of the following conditions: fatigue inflammation, appetite, bodyweight, body fat, blood lipid level, blood triglyceride level, bloodcholesterol level, inflammation and immunity in an individual comprisingadministering a pharmaceutically effective dose of a biologically activepeptide, said biologically active peptide having an amino acid sequencecomprising, consisting essentially of, or consisting of an amino acidsequence selected from the group consisting of SEQ ID No. 3, 7 or 21 orfunctional derivatives thereof.

A further aspect of the present invention is directed to a nutritionalcomposition containing the peptide comprising, consisting essentially ofor consisting of an amino acid selected from the group consisting of SEQID Nos. 4, 5, 8-11 or functional derivatives thereof and the use of thesame for the manufacture of a nutritional supplement.

In a further aspect of the present invention, enhanced derivatives ofthe peptide of SEQ ID Nos. 4, 5, 8-11 and functional derivatives thereofare provided. Enhanced derivatives of these peptides comprise anenhancement molecule operably linked to these peptides in such a manneras to improve or augment the therapeutic effectiveness of the peptide.The enhancement effect may be that of a prolonged effect, a shortenedeffect, a delayed onset of effect, a hastened onset of effect, anincreased intensity of effect, a decreased intensity of effect, areduction in side effects, the creation of one or more effects, adelayed subsiding of effect, a hastened subsiding of effect and atargeting of the peptide to a discrete location within an individual.Examples of such enhancement molecules and enhanced derivatives aredescribed below. In some aspects of the invention, the enhancedmolecules can treat or prevent, but are not limited to treating orpreventing, viral infections and immunological disorders. Additionalaspects of the present invention include methods of enhancing thetherapeutic effects of a peptide comprising, consisting essentially ofor consisting of a peptide selected from the group consisting of SEQ IDNos. 4, 5, 8-11 and their derivatives, comprising operably linking saidpeptide to a molecule which enhances the therapeutic effect. In someaspects of the invention, said operably linked molecule which enhancesthe therapeutic effect is not a peptide that is adjacent to a peptideselected from the group consisting of SEQ ID Nos. 4, 5, 8-11 and theirderivatives in a naturally occurring peptide.

In a further aspect of the invention, a composition comprising asubstantially pure peptide comprising, consisting essentially of, orconsisting of an amino acid sequence selected from the group consistingof SEQ ID Nos. 4, 5, 8-11 is provided.

In an additional aspect of the invention, a method of reducing theeffects of a human disease is provided, by administering apharmaceutically effective dose of a biologically active peptidecomprising, consisting essentially of, or consisting of an amino acidsequence selected from the group consisting of SEQ ID Nos. 4, 5, 8-11.In some embodiments, the human suffers from at least one of thefollowing conditions: fatigue, nutritional disorders, metabolicdisorders, lipid metabolism disorder, acidosis, immune activity, effectsof radiation, hepatitis, rejection of transplanted organ, the growth ofcancer and appetite.

In a further aspect of the invention, a method of modulating or treatinga condition comprising administering a biologically active peptidehaving an amino acid sequence comprising, consisting essentially of, orconsisting of essentially of SEQ ID No. 1 or a functional derivativethereof is provided, wherein the condition is selected from the groupconsisting of: fatigue, liver glycogen storage level, blood lactic acidlevel and immune reaction of an individual.

In an additional aspect of the invention, a method of modulating liverglycogen storage level, blood lactic acid level or immune reaction of anindividual is provided, by administering a pharmaceutically effectivedose of a biologically active peptide comprising, consisting essentiallyof, or consisting of an amino acid sequence comprising, consistingessentially of, or consisting of essentially of SEQ ID No. 1 or afunctional derivatives thereof.

In a yet further aspect of the invention, a biologically active peptideis provided, having an amino acid sequence comprising, consistingessentially of, or consisting of an amino acid sequence selected fromthe group consisting of SEQ ID No. 2 or 15 or functional derivativesthereof in the manufacture of a medicament for the treatment ofhepatitis.

In a yet further aspect of the invention, a method of treating hepatitisin an individual is provided, by administering a pharmaceuticallyeffective dose of a biologically active peptide, the biologically activepeptide having an amino acid sequence comprising, consisting essentiallyof, or consisting of an amino acid sequence selected from the groupconsisting of SEQ ID No. 2 or 15 or functional derivatives thereof.

In an additional aspect of the invention, a biologically active peptideis provided, having an amino acid sequence comprising, consistingessentially of, or consisting of SEQ ID No. 3 or functional derivativesthereof in the manufacture of a medicament for the alleviation offatigue.

In a further aspect of the invention, a method of alleviating fatigue inan individual is provided, by administering a pharmaceutically effectivedose of a biologically active peptide having an amino acid sequencecomprising, consisting essentially of, or consisting of SEQ ID No. 3 orfunctional derivatives thereof.

In a yet further aspect of the invention, the use of a biologicallyactive peptide is provided, having an amino acid sequence comprising,consisting essentially of, or consisting of SEQ ID No. 21 or functionalderivatives thereof in the manufacture of a medicament for modulating atleast one of the following conditions: inflammation, appetite, bodyweight, body fat, blood lipid level, blood triglyceride level, bloodcholesterol level, inflammation and immunity.

In a yet further aspect of the invention, a method of modulating atleast one of the following conditions is provided: inflammation,appetite, body weight, body fat, blood lipid level, blood triglyceridelevel, blood cholesterol level, inflammation and immunity in anindividual comprising administering a pharmaceutically effective dose ofa biologically active peptide, the biologically active peptide having anamino acid sequence comprising, consisting essentially of, or consistingof SEQ ID No. 21 or functional derivatives thereof.

In a further aspect of the invention, the use of a biologically activepeptide is provided, having an amino acid sequence comprising,consisting essentially of, or consisting of SEQ ID No. 7 or functionalderivatives thereof in the manufacture of a medicament for modulatingthe immune system of an individual.

In a yet further aspect of the invention, a method of modulating theimmune system of an individual is provided, by administering apharmaceutically effective dose of a biologically active peptide havingan amino acid sequence comprising, consisting essentially of, orconsisting of SEQ ID No. 7 or functional derivatives thereof.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As described herein, peptides described herein have been found to haveimportant biological activities. The peptides can be readily synthesizedby standard synthetic methods from L-amino acids, but may also besynthesized by genetic engineering methods using nucleic acids that havesequences encoding the individual peptides. All references cited areincorporated herein in their entirety.

As used herein, “substantially pure peptide” refers to peptides that areat least 40% and more preferably 60% and more preferably more than 90%pure. In the most preferred embodiment, the purity is about 99%-100%.The substantially pure peptide can be used to prepare pharmaceutical andnutritional formulations that may be complex mixtures as describedbelow.

As used herein, the term “hybrid peptide” is used to refer to peptidesthat contain additional peptides inserted into the original biologicallyactive peptide described herein having the sequence specified above orits functional derivatives, but still retain substantially similaractivity. The additional peptides include leader peptides that contain,for example, an amino acid sequence that is recognized by one or moreprokaryotic or eukaryotic cells as a signal for secretion of the hybridprotein to the exterior of the cell. The secretion may be a directsecretion, or indirectly through secretory vesicles.

As described above, another embodiment of the present invention is apeptide or polypeptide consisting essentially of a peptide of thepresent invention. As used herein, the terminology “consistingessentially of” refers to a peptide or polypeptide which includes theamino acid sequence of the peptides of the present invention along withadditional amino acids at the carboxyl and/or amino terminal ends andwhich maintains the activity of the peptides of the present inventionprovided herein. Thus, as a non-limiting example, where the activity ofthe peptide of the present invention is to modulate immune activity, apeptide or polypeptide “consisting essentially of” the peptide of thepresent invention will possess the activity of modulating immuneactivity as provided herein with respect to that peptide and will notpossess any characteristics which materially reduces the ability of thepeptide or polypeptide to modulate immune activity or which constitutesa material change to the basic and novel characteristics of the peptideas a modulator of immune activity. Thus, in the foregoing example, afull length naturally occurring polypeptide which has a primary activityother than modulating immune activity and which contains the amino acidsequence of a peptide of the present invention somewhere therein wouldnot constitute a peptide or polypeptide “consisting essentially of” apeptide of the present invention. Likewise, in the foregoing example, agenetically engineered peptide or polypeptide which has a primaryactivity other than modulating immune activity but includes the aminoacid sequence of a peptide of the present invention somewhere thereinwould not constitute a peptide or polypeptide “consisting essentiallyof” a peptide of the present invention.

In the preferred embodiment, the terminology “consisting essentially of”refer to peptides or polypeptides which have 4 or less amino acids inaddition to one of the peptide of the present invention. In the morepreferred embodiment, the same terminology refers to peptides orpolypeptides with 2 amino acids in addition to one of the peptide of thepresent invention. In the most preferred embodiment, the sameterminology refers to peptides or polypeptides with one amino acid inaddition to one of the peptide of the present invention.

Besides the example of immune activity modulation used for illustrationabove, the foregoing definition also applies to all the peptides of thepresent invention with respect to the activities provided for suchpeptides. In particular, the foregoing definition applies to peptides ofthe invention having activities in modulating fatigue, nutritionaldisorders, metabolic disorders, lipid metabolism disorder, acidosis,immune activity, effects of radiation, hepatitis, rejection oftransplanted organ, the growth of cancer and appetite.

Those skilled in the art can readily determine whether a peptide orpolypeptide consists essentially of a peptide of the present inventionunder the foregoing definitions by measuring the activity of the peptideor polypeptide using the assays as described below.

Those skilled in the art can readily determine whether a peptide orpolypeptide consists essentially of a peptide of the present inventionunder the foregoing definitions by measuring the activity of the peptideor polypeptide using the assays for modulation of disorders such asfatigue, nutritional disorders, metabolic disorders, lipid metabolismdisorder, acidosis, immune activity, effects of radiation, hepatitis,rejection of transplanted organ, the growth of cancer and appetite whichare provided herein with respect to a particular peptide of the presentinvention.

The terminology “disorder” as used herein and in the claims refers toany conditions in the body that fall outside the normal range asmeasured by standard methods. For example, in humans, “lipid metabolismdisorder” refer to a condition in which any of the lipid levels in thebody exceeds the normal range for that particular person based on thegender, height and age, as determined by standard medical diagnostictests.

The peptide may be administered by any suitable route. Exampleadministration routes are by intravenous injection, intramuscularinjection, intraperitoneal injection, subcutaneous injection, andsubcutaneous implantation, with or without delivery facilitating devicesuch as liposome, sustain release protection, and the like. Thus thepresent invention includes devices for injecting the peptide into asubject. In some embodiments the device may be a syringe. The peptidemay also be administered in any form of oral administration. Examplesmay include but are not limited to like tablet, capsule, suspension,solution, a lozenge, and the like, in the usual form withoutmodification or in a slow release form, or with or withoutgastro-enteric protection. The peptide may further be applied in anyform of topical application such as an ointment, cream, gel etc with orwithout transdermal facilitating device, or as inhalant of powder,dissolved, or as liposome protected form.

It is understood that it may be possible to add additional amino acidsto the amino or carboxyl termini of the disclosed peptides describedherein as another method of practicing the present invention. Forexample, one or two amino acids may be added to the disclosed peptidewithout affecting its biological function. It may also be possible toadd three or four amino acids and still maintain the function of thepeptides. These are all referred to as variants of the same peptide.Alternatively, one or two amino acids may be deleted from the peptidewithout affecting its biological activity. It may further be possiblefor three or four amino acids to be deleted without affecting thebiological function of the peptides. These are referred to as fragmentsof the instant peptide. Furthermore, derivatives of the peptide such asconservative replacement of one amino acid for another within the samefunctional class may be used to practise another aspect of the presentinvention. For example, peptides having non-polar or hydrophobic sidechains may be possible to substitute one side group for another withoutreducing biological activity. As a further example, a linker/spacer maybe inserted into the peptide to form variants, but the variants stillretaining its active moiety as the original peptide used in this study.These are also considered variants of the peptides. A peptide analogueas used herein, includes peptides that have amino acid molecules whichmimic the structure of the natural amino acid e.g. an analog with adifferent backbone structure, or D-amino acid substitution. As a furtherexample, although the amino acids used for synthesizing the peptides arein their L optical isomeric form, peptides with one or more of the aminoacids in the sequence substituted with the D-form may have similarbiological activities. The term “functional derivative” as used in theclaims is meant to include fragments, variants, analogues or chemicalderivatives of the peptide.

The use of the above-identified peptides in pharmaceutical formulationsmay be employed as possible treatment for disorders described herein,such as immunological disorders, cancer, fatigue, transplant rejection,and the like. The formulations may contain one of the identifiedpeptides mixed with other active or inactive constituents, includingother peptides. Alternatively, one of the listed peptides may be used toprepare the formulation together with peptides not listed here. They canbe administered in the form of intravenous, intramuscular,intracutaneous, subcutaneous or intradermal. The mode of administrationmay also be intra-arterial injection that leads directly to the organ inneed of treatment. Other modes of administration are transdermal,inhalation as powder or spray, and other forms of delivery known by onein the art. The formulation may also be orally taken, and may containcarriers that can be used to prevent gastric digestion of the peptideafter oral intake or any other carriers known in the art.

The pharmaceutical formulation may include any of the knownpharmaceutical carriers. Examples of suitable carriers include any ofthe standard pharmaceutically accepted carrier known to those skilled inthe art. These include but are not limited to, physiological salinesolution, water, emulsions including oil and water mixtures ortriglyceride emulsions, and other types of agents, fillers, coatedtablets and capsules. The appropriate carrier may be selected based onthe mode of administration of the pharmaceutical composition.

In a further aspect of the present invention, enhanced derivatives ofthe peptide described herein and functional derivatives thereof areprovided. The biologically active peptides of the present invention maybe conjugated to other biologically effective or useful molecules toprovide an additional effect or use or to enhance their therapeuticeffectiveness. Many potential conjugating molecules, their biologicaleffects and the methods for conjugation of the molecules to peptides areknown in the art. Exemplary conjugating molecules include but are notlimited to an organic compound, a carbohydrate, a sugar, apolysaccharide, an amino acid, an amino acid polymer, a peptide, asteroid, a protein, an isolated domain of a protein, a lipid molecule, afatty acid, a bile acid, a polyamine, a protease inhibitor, and thelike. A combination of conjugating molecules can also be used.

Some of the peptides of the invention have distinct therapeutic effectson a particular cell or tissue type. One important objective ofconjugating molecules to peptide drugs is the targeting of the peptideto a particular location or compartment within the body of an individualbeing treated. In this way, the peptide drug and its effects can beconcentrated at the location of the cell or tissue type on which it hasthe intended therapeutic effect. This can augment the effect that asimilar molar amount of the free, unconjugated peptide would have. Otherobjectives of conjugating molecules can include, for example, extendingthe life of the peptide, altering the solubility of the peptide,altering the activity of the peptide, and altering the bioavailabilityof the peptide.

Chemical reactions for conjugating the instant peptides to theconjugating partners can be deduced by one skilled in the art withoutundue experimentation. Various conjugation techniques are described, forexample, in Haas et al., Kidney Intl., 52(6):1693, 1997; Fiume et al.,Ital J Gastroenterol Hepatol, 29(3):275, 1997; Di Stefano et al.,Biochem. Pharmacol., 61(4):459, 2001; Huang et al., Chem. Biol.,7(7):453, 2000; Leopold et al., J. Pharmacokinet. Biopharm., 23(4):397,1995; Patel et al., Bioconjugate Chem., 8(3):434, 1997; Kramer et al. J.Biol. Chem., 269(14): 10621, 1994; Toth et al. (J. Med. Chem.,42(19):4010, 1999; Kim et al., (Biomaterials, 23:2311, 2002; Oldham etal. (Int. J. Oncology, 16:125, 2000; and Fitzpatrick et al. AnticancerDrug Design, 10:1, 1995, all of which are incorporated by referenceherein in their entireties.

Gene Therapy and Method of Treatment

Gene therapy based on the discovered peptide sequences can be performedby designing a nucleic acid sequence that code for one of thesepeptides. The nucleic acid may be synthesized chemically and operablyligated to a promoter, and cloned into an expression vector. Theexpression vector is then administered into the human body as the formof gene therapy for expression in the human cell. The term “geneticvectors” as used herein includes these expression vectors. Vectors thatcan be used for gene therapy includes adeno-associated virus (Mizuno, M.et al. (1998), Jpn J Cancer Res 89, 76-80), LNSX vectors (Miller, A. D.et al. (1993) Methods Enzymol 217, 581-599) and lentivirus (Goldman, M.J. et al. (1997) Hum Gene Ther 8, 2261-2268), the disclosures of whichare incorporated herein by reference in their entireties.

Other vehicles for peptide delivery include expression vectors encodingthe desired peptide that can be transferred into an organism which canreplicate in the host organism to which it is desired to administer thepeptide without significant detrimental effects on the health of thehost organism. For example, the expression vectors may be transferredinto an organism which is not pathogenic to the host organism to whichit is desired to administer the peptide. In some embodiments theexpression vector produces the desired peptide in a bacterial or fungalorganism which does not have significant detrimental effects on thehealth of the host organism to which the peptide is to be administered.For example, the expression vector encoding the desired peptide may bean expression vector which produces the desired peptide in an organismsuch as lactic acid bacteria, E. coli, or yeast. In one embodiment, theexpression vector produces the desired peptide in a microbe normallyfound in the mammalian gut or a microbe tolerated by the mammaliandigestive tract. Some of the microbial species in which the desiredpeptide can be expressed include, but are not limited to, Lactobacillusspecies, such as L. acidophilus, L. amylovorus, L. casei, L. crispatus,L. gallinarum, L. gasseri, L. johnsonii, L. paracasei, L. plantarum, L.reuteri, L. rhamnosus or others; Bifidobacterium species, such as B.adolescentis, B. animalus, B. bifidum, B. breve, B. infantis, B. lactis,B. longum or others; Enterococcus faecalis or Ent. facium;Sporolactobacillus inulinus; Bacillus subtilis or Bacillus cereus;Escherichia coli; Propionibacterium freudenreichii; or Saccharomycescerevisiae or Saccharomyces boulardii.

Nucleic acid sequences that encode the peptides of the presentinvention, chemically synthesized or produced by other means, includingbut not limited to the reverse transcription of mRNA to produce cDNAmolecules, can be incorporated into expression vectors for gene transferinto the desired organisms by methods of genetic engineering familiar tothose of skill in the art. The expression vectors may be DNA vectors orRNA vectors. For example, the expression vectors can be based on plasmidor viral genetic elements. The expression vectors can be vectors whichreplicate extra-chromosomally or vectors which integrate into thechromosome.

The expression vectors comprise a promoter operably linked to a nucleicacid encoding a peptide of the present invention. The promoter may be aregulatable promoter, such as an inducible promoter, or a constitutivepromoter. In some embodiments, the promoter may be selected to provide adesired level of peptide expression. In addition, if desired, theexpression vectors may comprise other sequences to promote theproduction, presentation and/or secretion of peptides. In someembodiments a nucleic acid encoding a peptide of the present inventionis operably linked to a nucleic acid sequence which directs thesecretion of the peptide. For example, the nucleic acid encoding thepeptide of the present invention may be operably linked to a nucleicacid encoding a signal peptide.

In some embodiments, the expression vectors which are engineered toencode the peptides of the present invention may be expression vectorswhich are adapted for expressing the peptide of the present invention ina bacterial species that makes up the normal gut flora of mammals, suchas Lactobacillus species and Bacillus subtilis Examples of suchexpression vectors can be found in U.S. Pat. No. 6,100,388, to Casas,and No. 5,728,571, to Bellini, respectively. These documents are herebyexpressly incorporated by reference in their entireties. It will beappreciated that any expression vector which facilitates the expressionof a peptide of the present invention in an organism which is notdetrimental to the health of the host organism to which the peptide isto be administered may be used.

In some embodiments, the expression vectors which are engineered toencode the peptides of the present invention may be expression vectorswhich are adapted for expressing the peptide of the present invention ina yeast species that is well tolerated by the mammalian gut, such asSaccharomyces cerevisiae; or, preferably, Saccharomyces boulardii, whichcan colonize the human gut and is used to treat certain forms ofdiarrhea. Yeast expression vectors can be used that constitutivelyexpress heterologous proteins and peptides, are highly stable, thus arewell transmitted to progeny cells during mitosis and meiosis and maycomprise coding sequence for a signal peptide or peptides that directhigh levels of recombinant protein secretion. An example of such a yeastvector is given in U.S. Pat. No. 6,391,585, to Jang et al., which ishereby expressly incorporated by reference in its entirety.

The expression vectors encoding the peptides of the present inventionmay be introduced into the organism in which it is intended to expressthe peptides through techniques known in the art. These techniquesinclude traditional methods of transforming bacteria, yeast, or othermicrobes, through the use of chemically competent bacterial cells,electroporation or lithium acetate transformation (for yeast), forexample, as well as recent advances in the transformation of bacterialspecies recalcitrant to these procedures. In some embodiments, theexpression vectors are introduced into lactic acid bacteria known to berecalcitrant to transformation using the method disclosed by Leer et al.(WO 95/35389), the disclosure of which is incorporated herein byreference in its entirety. The introduced sequences may be incorporatedinto microbial chromosomal DNA or may remain as extrachromosomal DNAelements.

This genetically engineered microbe containing the expression vector canthen be inoculated into the alimentary canal, vagina, trachea, etc., toachieve sustained immuno-therapy. In some embodiments, the organismsexpressing the peptides of the present invention are ingested in aninactive form or, preferably, in live form. In the gut thesemicroorganisms produce said peptides, release them into the lumen bysecretion or by lysis of the microorganism or otherwise present thepeptides to the host, whereby the peptides produce their intended effectupon the host organism. In other embodiments, peptides are presented tothe host at the mucous membrane of the nasal passages, vagina or thesmall intestine.

Another method of the treatment is the use of liposomes as a means fordelivering the specific nucleic acid to the cells in the human body. Thenucleic acid (such as an expression vector containing a nucleic sequencethat encodes peptides described herein can be delivered in anenvironment that encourages cellular uptake and chromosomalincorporation as described in Gao, X. and Huang, L. (1995) Gene Ther 2,710-722 and U.S. Pat. No. 6,207,456, the disclosure of which isincorporated herein by reference in its entirety. Alternatively, thepeptide itself can be encapsulated in the liposome and delivereddirectly, using a method described in U.S. Pat. No. 6,245,427, thedisclosure of which is incorporated herein by reference in its entirety.All the scientific publications and patents indicated are incorporatedherein by reference.

The nucleic acid sequences useful for the above-mentioned gene therapyand method of treatment include sequences that code for these peptidesand functional derivatives thereof. Any one of the numerous nucleic acidsequences may be used to code for these peptides and their derivativesbased on the degenerate codon system.

Example 1 The Effects of CMS-001 and CMS-0080N Exercise-Induced Fatigueof Mice

Objective:

To investigate the anti-fatigue effects of CMS-001 and CMS-008 on BALB/cmice.

Methods:

BALB/c mice exhaustive swimming model was used to observe theanti-fatigue effects of CMS-001 and CMS-008. The exhaustive swimmingtime, liver glycogen, serum urea nitrogen (BUN), and blood lactic acidlevel was observed after the administration of CMS-001 and CMS-008. Theserum malondialehyde (MDA), superoxide dismutase (SOD), alanineaminotransferase (ALT), and aspartate aminotransferase (AST) levels werealso observed to investigate the possible anti-fatigue mechanism ofCMS-001 and CMS-008.

Results:

CMS-001 and CMS-008 at suitable concentrations, were found to be able tolengthen the exhaustive swimming time of mice, reduce the BUN level,increase the liver glycogen level, and decrease the accumulation ofblood lactic acid, with statistical significance compared with thesaline control (P<0.05). It was also found that CMS-001 and CMS-008 werealso able to decrease the elevation of blood MDA, ALT, and AST levelsand increase the activity of SOD during exhaustive exercise, withstatistical significance compared with the saline control (P<0.05).

Conclusion:

CMS-001 and CMS-008 were found to have anti-fatigue properties and maybe used in the management of fatigue related disorders.

1 Material and Methods: 1.1 Drug and Reagents

CMS-001 and CMS-008 were custom synthesized by Shenzhen KangzhePharmaceutical Co. Ltd., Shenzhen, PR China.

Urea Nitrogen reagent kit: Beckman Coulter, Inc., Fullerton, Calif.,USA.

Anthracene Ketone reagent kit: Shanghai Fifth Union Laboratory,Shanghai, China, PRC.

MDA and SOD reagent kits: Nanjing Jiancheng Bio-engineering Corporation,Nanjing, China, PRC.

AST and ALT reagent kits: Beijing Zhongsheng Bio-engineering HighTechnology Corporation, Beijing, PRC.

1.2 Animals

Male BALB/c mice, specific pathogen free (SPF) grade, weighing 18-22 gwere obtained from Academy of Military Medicines and SciencesExperimental Animal Center, PR China.

The mice were randomized into groups of CMS-001 (20 μg/kg/day, 5μg/kg/day), CMS-008 (500 μg/kg/day, 125 μg/kg/day), and saline control.The test substance was administered by intraperitoneal injection onceper day for 30 continuous days. A week after the start the of the testsubstance administration, the mice were trained to swim for 10 min, atwater temperature of 25±1° C.

1.3 Experimental Equipment

Swimming tank (50 cm×50 cm×40 cm).

Fully automated Biochemistry Analyser RA-1000.

Lactic acid automated analyser 1500SPORT.

1.4 The Effect of CMS-001 and CMS-008 on the Exhaustive Swimming Time ofMice^([1])

30 min after the last injection, the mice were weighed, and lead piecescorresponding to 5% of the body weight were wrapped to the tails of themice at about 1 cm away from the body. The mice were placed in theswimming tank, at water depth of 30 cm and temperature of 25±1° C. Thelimbs of the mice were kept moving in the whole process. The time ofswimming until the death was recorded.

1.5 The Effect of CMS-001 and CMS-008 on Serum Urea Nitrogen Level afterExercise

30 min after the last injection, the mice were placed to swim in theswimming tank without addition of weight for 90 min, at water depth of30 cm temperature of 30±1° C. The mice were then allowed to rest for 30min, and blood was collected from the orbital sinus. The serum wasisolated and the BUN was determined by a fully automated BiochemistryAnalyser.

1.6 The Effect of CMS-001 and CMS-008 on Liver Glycogen Level ofMice^([2])

30 min after the last injection, the mice were killed by cervicaldislocation and the liver was dissected, washed with saline, and blotteddry with filter paper. About 100 mg liver was accurately weighed and theliver glycogen level was determined by the Anthracene Ketone reagentkit.

Glycogen weight (mg) in every 100 g of liver=DU/DS×0.5×homogenizationliquid vol./liver weight(g)×100×0.9.

Whereas:

DU=absorbency of sample

DS=absorbency of standard

Homogenization liquid vol=8 ml

1.7 The Effect of CMS-001 and CMS-008 on Blood Lactic Acid Level of Miceafter Exercise^([3])

30 min after the last injection, 20 μl of blood was first collected fromthe orbital sinus. The mice were then weighed, and lead piecescorresponding to 4% of the body weight were wrapped to the tails of themice at about 1 cm away from the body. The mice were placed to swim inthe swimming tank for 10 min, at water depth of 30 cm and temperature of30±1° C. The limbs of the mice were kept moving in the whole process.Immediately after the exercise and after resting for 20 min, 20 μl ofblood was collected from the orbital sinus again. The blood was addedinto 40 μl hypotonic buffer and sonicated. Lactic acid level in theblood was determined by the lactic acid analyzer.

The amount of lactic acid increase after exercise is calculated based onthe amount of lactic acid level immediately after exercise minus thelactic acid level before exercise.

The amount of lactic acid elimination after rest was calculated based onthe amount of lactic acid level immediately after exercise minus thelactic acid level after rest.

1.8 The Effect of CMS-001 and CMS-008 on the Serum SOD, MDA, AST, andALT Levels after Exhaustive Exercise

30 min after the last injection, the mice were placed to swim in theswimming tank without addition of weight at water depth of 30 cm andtemperature of 25±1° C.^([4]). By the first time the mice sunk, the micewere rescued and blood was collected from the orbital sinus immediately.The serum was isolated and the MDA, SOD, AST, and ALT levels determined.

1.9 Statistical Method

Difference between groups were analyzed by ANOV Analysis of variance.

2 Results

TABLE 1.1 The effect of CMS-001 and CMS-008 on the exhaustive swimmingtime of mice Dosages Swimming time Swimming-time groups (μg/kg/day) No.(min) extension (%) CMS-001 20 15  99.8 ± 30.6* 48.0* CMS-001 5 18 105.7± 19.8* 56.7* CMS-008 500 15 116.5 ± 17.7* 72.7* CMS-008 125 18 106.5 ±19.4* 57.9* Saline — 16 67.5 ± 28.0 *compared with the saline control, P< 0.05

TABLE 1.2 The effect of CMS-001 and CMS-008 on serum urea nitrogen levelafter exercise groups Dosages (μg/kg/day) No. Urea Nitrogen (mmol/l)CMS-001 20 17 7.3 ± 2.1* CMS-001 5 18 6.9 ± 1.6* CMS-008 500 17 7.1 ±2.0* CMS-008 125 18 7.2 ± 2.3* Saline — 17 9.0 ± 2.5 *compared with thesaline control, P < 0.05

TABLE 1.3 The effect of CMS-001 and CMS-008 on liver glycogen level ofmice Dosages liver glycogen (mg/100 g liver groups (μg/kg/day) No.tissue) CMS-001 20 8 1567 ± 604* CMS-001 5 10  439 ± 271* CMS-008 500 81408 ± 457* CMS-008 125 10 1275 ± 726* Saline — 10 271 ± 217 *comparedwith the saline control, P < 0.05

TABLE 1.4 The effect of CMS-001 and CMS-008 on blood lactic acid levelof mice after exercise Lactic acid increase Lactic acid Dosages afterexercise elimination groups (μg/kg/day) No. (mmol/l) after rest (mmol/l)CMS-001 20 15 2.56 ± 0.41* 3.14 ± 0.64* CMS-001 5 13 2.60 ± 0.33* 2.96 ±0.33* CMS-008 500 15 2.23 ± 0.30* 3.71 ± 0.22* CMS-008 125 13 2.70 ±0.15* 3.62 ± 0.57* Saline — 14 4.60 ± 0.16  2.00 ± 0.44  *compared withthe saline group P < 0.05

TABLE 1.5 The effect of CMS-001 and CMS-008 on the serum SOD, MDA, AST,and ALT levels after exhaustive exercise Dosages MDA SOD AST ALT groups(μg/kg/day) No. (nmol/l) (U/ml) (U/L) (U/L) CMS-001 20 9 4.4 ± 0.2*400.6 ± 45.6* 117.0 ± 19.2* 32.7 ± 3.0* CMS-008 500 9 4.1 ± 0.4* 415.7 ±31.5* 116.6 ± 18.8* 32.9 ± 5.2* Saline — 9 6.9 ± 0.3 342.7 ± 27.3 145.2± 32.3 40.2 ± 8.2 *compared with the saline group P < 0.05

Conclusion

This study showed that CMS-001 and CMS-008 have the followingproperties:

1. Extend the swimming time of the mice, showing that CMS-001 andCMS-008 can increase the exercise capacity of the animal^([5]).

2. Decrease the BUN of the mice, showing that CMS-001 and CMS-008 candecrease the necessity for catabolism of protein for energy productionduring exercise^([6]).

3. Increase liver glycogen reserve of the animal during rest, giving theanimals increased capacity for exercise^([6]).

4. Decrease the blood lactic acid level after exercise, and increase thespeed of lactic acid elimination thereafter, showing that CMS-001 andCMS-008 can decrease the speed of lactic acid production or lactic acidelimination during exercise^([7]).

5. Decrease the MDA level, showing that CMS-001 and CMS-008 can decreasefree radical production during exercise^([8]).

6. Increase the SOD level, showing that CMS-001 and CMS-008 can increasefree radical elimination during exercise^([8]).

7. Decrease ALT and AST level, showing that CMS-001 and CMS-008 canprotect heart and liver cellular damage during exercise.

8. CMS-001 and CMS-008 may be useful in the management of exerciserelated fatigue disorders.

References:

The following references are incorporated herein by reference in theirentireties.

-   1. Mizunoya W, Oyaizu S, Ishihara K, et al. Protocol for measuring    the endurance capacity of mice in an adjustable-current swimming    pool. Biosci Biotechnol Biochem. 2002 May; 66(5):1133-6.-   2. HE Ling, WANG Ming, CHEN Run etc. The effect of blood lactic    acid-blood serum carbamide nitrogen and liver hepatin affected by    gen-seng. Prevent Medicine Literature Information, 2002,    8(3):293-294.-   3. WANF Xiao-xue, QIU Juan, SONG Yu etc. Study on Effect of Theanine    of Fatigue. China Commonality Sanitation Journal, 2002,    18(3):315-317.-   4. Thomas D P, Marshall K I. Effects of repeated exhaustive exercise    on myocardial subcellular membrane structure. Int J Sport Med, 1988,    (9):257-260.-   5. JIN Zong-lian. Evaluation principle and method of function food.    Beijing: Beijing University Publishing Company, 1995.-   6. Sanitation ministry, Evaluation progress and test methods of    health care food. Ministry of Public Health, PR China.-   7. Westerblad, et al. Changes of intracellular pH due to repetitive    stimulation of single fibers from mouse skeletal muscle. J Physiol;    1992 April; (499):49-71.-   8. Groussard C, Rannou-Bekono F, Machefer G, et al. Changes in blood    lipid peroxidation markers and antioxidants after a single sprint    anaerobic exercise. Eur J Appl Physiol. 2003 March; 89(1):14-20.

Example 2 Immuno-Regulatory Effects of CMS-0010N Radiotherapy InducedImmuno-Comprised Mice

Objective:

To observe the immuno-regulatory effects of CMS-001 on radiotherapyinduced immuno-compromised animal model.

Methods:

An immuno-compromised mice model was first set up by Cs¹³⁷ irradiation.CMS-001 was then applied and the change in T lymphocyte proliferationrate was then analyzed by the MTT method.

Results:

At 20 μg/kg/day, CMS-001 was found to be able to increase the rate of Tlymphocyte proliferation, with statistical significance (P<0.05).

1 Material and Methods 1.1 Drug and Reagents

CMS-001: Custom synthesized by Shenzhen Kangzhe Pharmaceutical Co. Ltd.,Shenzhen, PR China.

Hank's solution, Bovine fetal serum, and RPMI-1640: Hyclone, Logan,Utah.

ConA and MTT: Sigma Chemical Co., St. Louis, Mo., USA.

1.2 Experimental Animals

Male Balb/c mice, specific pathogen free grade (SPF), weighing 18-22 gwere obtained from Academy of Military Medicines and Sciencesexperimental animal center, PR China.

1.3 Preparation of Animal Model, Administration of CMS-001, andQuantitation of T Lymphocyte Proliferation Rate^([1])

Balb/c male mice were randomized into groups of: CMS-001 (20 μg/kg/day,5 μg/kg/day), the saline control, and the normal control. All miceexcept the normal control group were exposed to 600 rad Cs¹³⁷ (82.83rad/min for 7.2 min). The test substance was dissolved in 0.5 ml saline,and applied intraperitoneally once per day for 15 continuous days afterirradiation. On the day after the last test substance administration,the spleens of the mice were dissected aseptically. The spleens weredispersed to single cells, washed, and the cell concentration adjustedto 4×10⁶/ml with RPMI-1640. To a 96 wells culture plate, 100 μl cellsand 100 μl Con A (to final concentration of 5 μg/ml) were added perwell. Baseline blank controls without ConA were also prepared. The cellswere incubated at 37° C. and 5% CO₂ for 68 hrs. The proliferation of Tlymphocyte was determined by the MTT method^([2]). The stimulation indexwas calculated according to the following: Stimulate index (SI)=mean ODvalue of ConA wells/mean OD value of the baseline blank wells.

2 Result

TABLE 2.1 The effect of CMS-001 on T lymphocyte proliferation inradiation induced immuno-comprised mice. Groups dosage No. SI CMS-001 20μg/kg/day 10 1.27 ± 0.19* saline control — 8 1.11 ± 0.08  normal control— 10 3.42 ± 0.93* *compared with saline group P < 0.05

3 Conclusion

CMS-001 was found to be able to stimulate the T lymphocyte proliferationin radiation induced immuno-compromised mice, with statisticalsignificance, showing that CMS-001 may be used as an immuno-stimulant onimmuno-comprised patients after radiotherapy.

References:

The following references are incorporated herein by reference in theirentireties.

-   1. New drugs (Western medicine) research direction principle before    clinic. Chinese Sanitation Ministry Drug Political Situation. 1993,    7:128-137.-   2. Qiu Zhi-qiang. The influence of Fuzheng buxuegao to blood system    and immunity function affected by radiotherapy. Lanzhou Medical    Transaction, 2003, 3(28).

Example 3 Effect of CMS-0140N the Survival of Skin Allograft in Mice

Objective:

To investigate the immunosuppressive effect of CMS-014 by the skinallograft model.

Methods:

Pieces of tail skin from C₅₇BL/6 mice were transplanted to BALB/c miceand the mean survival time (MST) of the skin allograft was observed.

Results:

CMS-014 was found to be able to prolong the survival of the skinallograft, with statistical significance (p<0.05) compared with thesaline control.

Conclusion:

CMS-014 may be used as an immuno-suppressing agent to control therejection reaction after organ transplantation.

1 Materials 1.1 Drugs and Other Reagents

CMS-014: Custom synthesized by Shenzhen Kangzhe Pharmaceutical Co. Ltd.,Shenzhen, PR China.

Cyclosporine A (CsA): Novartis Pharmaceutical Co. Ltd., Basel,Switzerland.

Saline: China OTSUKA Pharmaceutical Co. Ltd., Tianjin, PR China.

Na₂S: Tianjin Beilian Chemical Co. Ltd., Tianjin, PR China.

1.2 Animals

Recipients: Balb/c(H-2^(d)) mice, specific pathogen free grade (SPF), 6weeks old, weighing 18-22 g: Military Medical Academy of Science, China.

Donors: C₅₇BL/6(H-2″) mice, SPF, 6 weeks old, weighing 18-22 g: MilitaryMedical Academy of Science, China.

2 Grouping and Treatment 2.1 Grouping and Test Substance Administration

Balb/c mice were randomized into groups of CMS-014 (500 μg/kg/day, 250μg/kg/day, 125 μg/kg/day), Cyclosporine A (10 mg/kg/day), and salinecontrol (0.5 ml/day). Half of the mice were male and half were female.

The test substances were dissolved in 0.5 ml saline andintraperitoneally injected for 5 days before the skin transplantation,followed by daily injection until the end point of rejection of the skingraft.

2.2 Skin Grafting

A patch of hair at the back of Balb/c mice was removed by 8% Na₂Ssolution. On the next day, a wound bed of approximately 1 cm² wasproduced by removing the skin surgically, and then a piece offull-thickness tail skin of 1 cm² from sex-matched donor C₅₇BL/6J micewas placed onto the wound bed. The surgical site was covered andprotected with a layer of paraffin gauze, and a covering plaster wasapplied. The plaster was removed 6 days after transplantation^([1]), andthe recipient mice were monitored daily for the viability of theallograft. The end point of rejection was taken as only less than 10% ofthe allograft remained viable^([2]).

2.3 Statistical Analysis

Statistical analysis was performed with Kaplan-Meier survival test.

3 Results

TABLE 3.1 Effect of CMS-014 on the MST of mice skin allografts GroupsDosages No. MST (days) CMS-014 500 μg/kg/day 9  9.8 ± 0.4* CMS-014 250μg/kg/day 10  9.8 ± 0.5* CMS-014 125 μg/kg/day 9 10.0 ± 0.5* CsA 10mg/kg/day 9 11.6 ± 0.8* saline 0.5 ml/day 8 8.1 ± 0.4 *Compared withsaline control group, p < 0.05

4 Conclusion

CMS-014 was found to be able to prolong the survival of the skinallograft, with statistical significance (p<0.05) compared with thesaline control. CMS-014 may be used as an immuno-suppressing agent tocontrol the rejection reaction after organ transplantation.

5 References

The following references are incorporated herein by reference in theirentireties.

-   [1] Ming Jiankuo, Wang Xingbing, Huang Baojun, et al. Peptide    Nucleic Acid Antisense Prolongs Skin Allograft Survival by Means of    Blockade of CXCR3 Expression Directing T Cells into Graft. The    Journal of Immunology, 2003, 170:1556-1565.-   [2] Steven H. Borenstein, Jeremy Graham, et al. CD8+ T Cells Are    Necessary for Recognition of Allelic, But Not Locus-Mismatched or    Xeno-, HLA Class I Transplantation Antigens. The Journal of    Immunology, 2000; 165:2341-2353.

Example 4 The Inhibition Effect of CMS-017 and CMS-0230N Hepatitis BVirus In Vitro Example 4a Human Hepatitis

Test peptides were added to the culture medium of 2.2.15 cell line.After incubation, the concentrations of hepatitis B surface antigen(HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B virus DNA(HBV-DNA) in the culture medium were determined and compared with thatof the control. Peptides CMS-017 and CMS-023 at suitable concentrationwere found to have anti-hepatitis B virus activity in vitro, withstatistical significance compared with the control (P<0.05). It isconcluded that these peptides may be used in the management of hepatitisB virus infection.

1 Materials

The peptides cms-017 and cms-023 were custom synthesized (of 1-aminoacids origin) by American Peptide Company, Inc., Sunnyvale, Calif., USA.

2.2.2.15 cell strain, transfected by human hepatitis B virus (HBV) DNA,was supplied by the National Center For Drug Screening (Shanghai, China)(test one) and the Department Of Infectious Diseases Of The FirstHospital affiliated to Beijing University (test two).

Cell culture medium MEM was from GIBCO®, Invitrogen, Carlsbad, Calif.,USA.

Elisa kits for HBsAg and HBeAg assays were from Shanghai Shiye KehuaBiotech. Company, Shanghai, PR China.

Fluorescence quantitative PCR kit for the determination of HBV-DNA wasfrom DA-AN gene company of Zhongshan Medical University, Guangzhou, PRChina.

2 Methods Test 1: Inhibitory Effect of Peptides on HBsAg and HBeAg atMaximum Non-Toxic Concentration

2.2.15 cells at log phase were harvested and adjusted to 2×10⁶/ml withMEM (containing 10% fetal bovine serum, 100 mg/ml penicillin, and 100U/ml streptomycin). The suspension was inoculated on a 24-well cultureplate with 1.5 ml per well, and incubated at 37° C., 5% CO₂ for 48 hoursfor attachment. The peptides were added to the final concentration of400 μg/ml with 3 parallel wells per sample. One set of blank control (3wells) was also prepared, in which the peptide was replaced with culturemedium. The plate was incubated at 37° C. for 4 days, the culture mediumwas then exchanged with freshly prepared medium (of the same compositionas the original), and the plate was incubated for a further 4 days. Bythe end of incubation, the supernatants of the culture were collected,and the titers of HBsAg and HBeAg were determined by ELISA methodaccording to the instruction of the detection kit manufacturer.

The inhibition percentages of the drugs were calculated as below:

% inhibition=(Average concentration of control−Average concentration ofsample)/Average concentration of control×100%

Test 2: Inhibitory Effect of Peptides on HBV-DNA

The preparation and incubation of cell suspension was repeated as intest 1 above, but at the peptide concentration of 160 μg/ml. By the endof incubation, the supernatants were collected and the HBV-DNAconcentrations were determination by fluorescence quantitative PCR,according to the instruction of the detection kit manufacturer.

% inhibition=(Average DNA concentration of control−Average DNAconcentration of sample)/Average DNA concentration of control×100%

Statistics

Student t test was used for the statistical analysis. P<0.05 was takenas statistically significant.

Results

TABLE 4.1 Inhibitory effect of peptides on HBsAg and HBeAg at 400 μg/mlPeptide Inhibition ratio to HBsA Inhibition ratio to HBeA CMS-017 68.6%*62.2%* CMS-023 58.4%* 53.7%* *Compared with the blank control, P < 0.05

TABLE 4.2 Inhibitory effect of peptides on HBV-DNA at 160 μg/ml Peptide% HBV-DNA inhibition CMS-017 90.8%* CMS-023 70.8%* *Compared with theblank control, P < 0.05.

Conclusion

At suitable concentration, CMS-017 and CMS-023 were found to be able toinhibit the development of hepatitis B virus in vitro, with statisticalsignificance compared with the control (P<0.05). This shows that CMS-017and CMS-023 may be used in the management of hepatitis B related viralinfections.

Example 4b The Antiviral Effects of CMS-017 on Duck Hepatitis B In Vivo

Objective:

To investigate the antiviral effects of CMS-017 on duck hepatitis Bvirus (DHBV) in vivo.

Methods

CMS-017 dissolved in saline was applied to Chongqing duck hepatitis Banimal model by intramuscular injection for 28 days. The serum levels ofDHBV DNA and DHBsAg (duck hepatitis B surface antigen) were observed byserum dot-blot hybridization and ELISA respectively and compared withblank control.

Results

CMS-017 was found to be able to reduce the serum levels of DHBV DNA andDHBsAg during the treatment period (P<0.01). Upon discontinuation oftreatment for 7 days, rebound was not observed.

Materials and Methods 1 Animal Model^([2])

0.1 ml DHBV DNA positive serum (5×10⁷ copies/ml) was inoculated into theabdominal cavity of one-day old Chongqing ducks to set up the hepatitisanimal model. 10 days after inoculation, blood samples were collectedfrom the external jugular vein and success of infection was confirmed bydot-blot hybridization with DHBV DNA probe labeled with digoxi^([3]).

The ducks were bred to 2 weeks old for entrance into the study.

2 Grouping and Administration

Ducks confirmed with DHBV infection were randomly divided into thefollowing groups:

1) Control group (n=12): Normal saline. 1 ml per day, once per day, andapplied by intramuscular injection.

2) CMS-017 group (n=12): 200 μg/kg/day CMS-017 (dissolved in 1 ml normalsaline) applied by intramuscular injection, once per day.

The treatment lasted for 4 weeks, and observation continued for anotherone week after termination of treatment. 1 ml blood samples were drawnfrom the external jugular vein on days 0, 7, 14, 21, 28, and 35 from thestart of treatment. The sera were isolated^([4]) and stored at −20° C.until analysis.

3 Parameters to be Monitored

1) DHBV DNA Serum Level

DHBV DNA probe was fluorescent-labeled according to the labeling kitprotocol from the manufacturer (Roche Co.). Duck sera were dot-blotted(2 dot per sample) onto nitrocellulose membrane and hybridized withfluorescent-labeled probe for DHBV DNA quantitation^([3]). DHBV DNA 40μl+DHBsAg 100 μl was used as internal standard. CDP-Star fluorimetryreagent was used for amplification of the fluorescence. Vuego Scan(Brisa-620st) scanner was used for film scanning, and Discovery SeriesQuantity One software for quantitative analysis of the blots. The blotvalue was described as “volume” (volume=intensity×mm²)

2) DHBsAg Serum Level

DHBsAg level was determined by ELISA method^([5-8]), and the O.D valuewas obtained by ELISA reader (Bio-TEK Co.) at 490 nm.

4 Statistical analysis

Paired t-test was carried out for each group using SPSS software.

Results

1. The Changes of the Serum DHBV DNA Concentration

TABLE 4.3 Serum DHBV DNA titer during pre- and post-treatment DHBV DNA(volume) Pre-treatment Week 1 Week 2 Week 3 Week 4 Week 5 Normal saline2055.9 ± 521.8 1952.5 ± 621.5 2048.6 ± 692.3 2031.2 ± 722.3 1886.0 ±641.3 2118.5 ± 468.1 CMS-017 2207.0 ± 237.5 1991.8 ± 378.9* 1925.0 ±549.9* 1743.9 ± 555.0* 1544.9 ± 389.7* 1742.7 ± 437.0* *Compared withsaline control P < 0.05

2. The Changes of the Serum DHBsAg Level

TABLE 4.4 Serum DHBsAg O.D. value during pre- and post-treatmentPre-treatment Week 1 Week 2 Week 3 Week 4 Week 5 Normal saline 0.949 ±0.688 0.761 ± 0.540 0.892 ± 0.762 0.867 ± 0.802 0.701 ± 0.673 0.871 ±0.634 CMS-017 0.928 ± 0.402 0.761 ± 0.328 0.668 ± 0.310* 0.551 ± 0.268*0.479 ± 0.279* 0.488 ± 0.182* *Compared with saline control P < 0.05

Conclusion

At suitable concentration, CMS-017 was found to have anti-hepatitisproperties in vivo, with statistical significance compared with thesaline control (P<0.05). CMS-017 may be used in the management of viralinfection related disorders.

References:

The following references are incorporated herein by reference in theirentireties.

-   1. Chen Yaxi, Guo shuhua, Zhang Dingfeng, et al. Foundation and    application of Chongqing duck hepatitis B model. Chinese Journal of    Hepatology. 1993; 1(2):89-91.-   2. Chen Yaxi, Guo shuhua, Chen Xuehua. Preparation and application    of DHBV DNA probe labeled with digoxin. Journal of Chongqing    University of Medical Sciences. 1994; 19(4):295-297.-   3. Tang Ni, Huang Ailong, Guo shuhua, et al. Systemic foundation and    application of serological parameters of humoral immunity to duck    hepatitis B virus. Chinese Journal of Hepatology. 2001; 9(1):13-15.-   4. Tang Ni, Huang Ailong, Guo shuhua, et al. Purification of Duck    hepatitis B surface antigen and its applications. Journal of    Chongqing University of Medical Sciences. 2001; 26(1):14-16.-   5. Tang Ni, Huang Ailong, Guo shuhua, et al. A comparison of    specifically immune response in DHBV-infected ducks. Chinese Journal    of Hepatology. 2001; 9(3):166-168.-   6. Tang Ni, Huang Ailong, Qi Zhenyuan, et al Immune response of    acutely infected ducks to duck hepatitis B virus. Chinese Journal of    Microbiology and Immunology 2000; 2(4):24-29.-   7. Chen Yaxi, Guo shuhua, Qi Zhenyuan, et al. An experimental study    of lamivudine against duck hepatitis B virus in combination with    famciclovir. Chinese Journal of Hepatology. 2001; 9(4):209-211.-   8. Qiu Zhi-qiang. The influence of Fuzheng buxuegao to blood system    and immunity function affected by radiotherapy. Lanzhou Medical    Transaction, 2003, 3 (28).

Example 5 The Inhibitory Effect of CMS024-160N Nude Mice-TransplantedHuman Gastric Carcinoma BGC-823 Xenograft 1 Materials 1.1 Drugs andReagents

CMS024-16: Custom synthesized by Shenzhen Kangzhe Pharmaceutical Co.Ltd., Shenzhen, PR China.

5-Fu: Tianjin Jinyao Amino Acid Co., Tianjin, PR China.

Fetal bovine serum (FBS): Hyclone, Logan, Utah.

RPMI-1640 cell culture medium: GIBCO®, Invitrogen, Carlsbad, Calif.,USA.

1.2 Animals

Healthy female BALB/c (nu/nu) nude mice, specific pathogen free grade(SPF), 4-5 weeks old: Academy of Military Medical Sciences, China.

1.3 Cell Lines

Human gastric carcinoma cell line BGC-823: Cancer Research Department,China Medical Science Institute.

2 Methods 2.1 Animal Model Preparation and Determination of Anti-TumorEffects^([1])

Hunan gastric carcinoma cell line BGC-823 at log phase were adjusted tothe concentration of 2×10⁷/ml and then inoculated subcutaneously to theback of nude mice (0.1 ml per mouse). The inoculated animals wererandomized into groups of CMS024-16 (160 μg/kg/day, 320 μg/kg/day, and640 μg/kg/day, all at 0.2 ml/day), positive control (5-Fu, 20 mg/kg/dayat 0.2 ml/day), and negative control (normal saline, 0.2 ml/day).Administration of test substances by intraperitoneal injection wasstarted on the next day after tumor implantation once per day for 30continuous days. On the next day after the last injection, the tumormasses were collected and their weights determined Inhibition rate=(Meanweight of negative control−Mean weight of test group)/(Mean weight ofnegative control)×100%

2.2 Statistical Analysis

Data were expressed as Mean±SD. ANOVA of the SPSS software was used forthe statistical analysis. P values <0.05 were taken as statisticallysignificant.

3. Results

TABLE 5.1 The inhibition effect of CMS024-16 on nude mice-transplantedhuman gastric carcinoma BGC-823 xenograft Inhibition rate Group Dosage NTumor weight (g) (%) CMS024-16 640 μg/Kg/day 8 1.31 ± 0.77* 54.4*CMS024-16 320 μg/Kg/day 8 1.59 ± 1.017* 44.7* CMS024-16 160 μg/Kg/day 81.32 ± 0.68* 54.0* 5-Fu  20 mg/Kg/day 9 1.70 ± 0.70* 40.8* Normal saline 0.2 ml/day 8 2.87 ± 1.05 — *Compared with normal saline group, p <0.05.

4. Conclusion

CMS024-16 at a dosage of 160-640 μg/kg/day, was found to be able toinhibit the growth of nude mice-transplanted human gastric carcinomaBGC-823 xenograft, with statistical significance compared with thenormal saline group (P<0.05).

Reference:

The following reference is incorporated herein by reference in itsentirety.

-   1. Li X H, Zhang G Y, Luo F J, Xu M H, Li Q. The effect of    Helicobacter pylori on the expression of metallo proteinases in    gastric carcinoma cell lines. World J Chinese Digestion, 2003,    11(5):544-546.

Example 6 The Inhibitory Effects of Peptides on Hepatocarcinoma H22 InVivo

Objective:

To investigate the effects of peptides on the growth of hepatocarcinomaH22 in mice.

Methods:

BALB/C mice were randomized into groups of saline control, 5-Fu, normalcontrol, peptides. Liver carcinoma H22 cells were transplanted byintraperitoneal injection and the test substances administered also byintraperitoneal injection once daily. The survival time of the mice wasrecorded and compared with the controls.

Results:

At doses of 80 μg/kg/day, CMS-024.04, CMS-024.05, CMS-024.14, CMS-024.16were found to be able to prolong the survival of mice transplanted withH22 tumor cells, with statistical significance compared with the salinecontrol group (P<0.05).

Conclusions: CMS-024.04, CMS-024.05, CMS-024.14, and CMS-024.16 werefound to be able to prolong the survival of mice with transplanted livercancer H22, showing that these peptides may be used for the managementof cancers.

1 Materials and Methods 1.1 Drugs and Reagents

CMS-024.04, CMS-024.05, CMS-024.14, and CMS-024.16 were custommanufactured by Shenzhen Kangzhe Pharmaceutical Co. Ltd., Shenzhen, PRChina.

5-FU (5-fluorouracil) was from Tianjin Jinyao Aminophenol Ltd., Tianjin,China.

Saline was from China OTSUKA Pharmaceutical Co. Ltd., Tianjin, China.

RPMI-1640 and Fetal bovine serum (FBS) were from GIBCO®, Invitrogen,Carlsbad, Calif., USA.

D-Hanks' solution were from Sigma Chemical Co., St. Louis, Mo., USA.

1.2 Animals

Healthy BALB/c mice (CLA grade, 6-8 weeks, weight 18-22 g) were from theAnimal Center of Military Medical Academy of Science, Beijing, China.

1.3 Cell Lines

Mice hosting hepatic carcinoma H22 cell strain were from TumorDepartment of Medical Institute of China Medical Academy of Science,Beijing, China.

1.4 Grouping of Animals

Healthy BALB/c mice were randomized into groups of CMS-024.04,CMS-025.05, CMS-024.14, and CMS-024.16 (80 μg/kg/day), 5-Fu (20mg/kg/day, once for every two days), saline (0.2 ml/day), and normalgroup (without tumor cell inoculation).

1.5 Administration of Hepatic Carcinoma H22 Mice Model

Mice inoculated with hepatocarcinomas for 6-8 days were sacrificed. Theascites was collected and the cell concentration adjusted to 5×10⁷/mlwith D-Hanks' solution. 0.2 ml of this was inoculated intraperitoneallyto each BALB/c mouse except for the normal controls.

1.6 Test Substance Administration

Administration of test substance was started on the next day after thetumor cell inoculation. The peptides and saline were applied daily and5-FU once every two days for 60 continuous days or until the death ofthe mice.

1.7 Survival Time Recording

The time of death was recorded and survival time prolongationcalculated. Survival time prolongation was calculated as below:

Survival prolongation (%)=(Average survival days of test group)−(Averagesurvival days of saline control group)/(Average survival days of salinegroup)×100%.

The mice that survived for more than 60 days were considered aslong-term survivors.

1.8 Statistical Method

Kaplan-Meier method was used for statistical comparison and P valuesequal to or less than 0.05 was taken as statistically significant.

2 Results

The experiment was done in two separate times, with results as below.

TABLE 6.1 Effect of peptides on the survival of mice transplanted withH22 hepatocarcinoma Survival Survival Survival time time ProlongationGroups Dosage N (days) (days) (%) CMS-024.04 80 μg/kg 16 19.0 ± 0.9 20.2± 4.4* 15.8* CMS-024.05 80 μg/kg 16 19.0 ± 1.0 19.3 ± 2.4* 10.4* Salinecontrol 0.2 ml/day 16 18.0 ± 0.3 17.4 ± 2.1 — 5-Fu 20 mg/kg 16 30.0 ±2.0 32.5 ± 6.3* 84.5* Normal — 16 60 60 — *Compared to saline controlgroup, P < 0.05

TABLE 6.2 Effect of peptides on the survival of mice transplanted withH22 hepatocarcinoma Survival Survival Survival time time ProlongationGroups Dosage N (days) (days) (%) CMS-024.14 80 μg/kg 16 16.0 ± 0.0 27.6± 8.5* 50.3 CMS-024.16 80 μg/kg 16 18.0 ± 1.0 29.0 ± 7.9* 57.8 Salinecontrol — 16 16.0 ± 0.1 18.4 ± 3.3 — 5-Fu 20 mg/kg 16 18.0 ± 0.4 24.5 ±11.1* 33.3 Normal — 16 60 60 — *Compared to saline control group, P <0.05

3 Conclusions

CMS-024.04, CMS-024.05, CMS-024.14, and CMS-024.16 were found to be ableto prolong the survival of mice with transplanted liver cancer H22, withstatistical significance compared with the saline control group, showingthat these peptides may be used for the management of cancers.

References:

The following references are incorporated herein by reference in theirentireties.

-   [1] Y Zhai and Z J Lu. Effect of thalidomide on tumor growth in    mouse hepatoma H22 model. Ai Zheng, December 2003; 22(12):1301-6.-   [2] Y X Yang, L Zhu, X He, et al. Antitumor activity of    mitoxantrone-nanosphere against murine liver tumor H22. Sichuan Da    Xue Xue Bao Yi Xue Ban, January 2004; 35(1):68-70.

Example 7 Effects of CMS-0300N Carrageenin Induced Foot Swelling in Rats

Objective:

To investigate the anti-inflammatory properties of CMS-030.

Methods:

Carrageenin induced foot swelling animal model was used to study theanti-inflammatory properties of CMS-030.

Results:

At the dosage of 2-20 μg/ml, CMS-030 was found to be able to suppressthe foot swelling in the treatment group compared to the control groups,with statistical significance (P<0.01).

Conclusion:

CMS-030 was shown to have a statistically significant anti-inflammatoryeffect on carrageenin induced foot swelling rat animal model.

1 Materials and Methods 1.1 Drugs and Reagents

CMS-030: Custom synthesized by Shenzhen Kangzhe Pharmaceutical Co. Ltd.,Shenzhen, PR China.

Carrageenin: Sigma Chemical Co., St. Louis, Mo., USA.

Dexamethasone (DXM): Tianjin JinYao Co. Ltd., Tianjin, PR China.

1.2 Animals

Wistar rats, 6-8 weeks old, weighing 180-220 g, Vitalriver ExperimentAnimal Co. Ltd., Beijing, PR China.

1.3 Methods^([1])

Wistar rats were divided randomly into DXM group (0.2 mg/kg/day), salinecontrol group (1 ml/day), and three dosages of CMS-030 groups (2,10,20μg/ml/day). All of the drugs were diluted to 0.5 ml with saline, andwere administered intraperitoneally (i.p) daily. Two weeks after drugadministration, 0.15 ml of 1% carrageenin saline solution was applied tothe right foot by subcutaneous injection. 0.5 hr later, thecircumference of right foot was measured precisely, and foot swellingwas calculated by subtracting the pre-challenge circumference from thepost-challenge value Inhibition index (%)=(foot swelling of salinecontrol group−foot swelling of drug group)/foot swelling of salinecontrol group×100%.

1.4 Statistical Analysis

Statistical analysis was performed with analysis of One-Way ANOVA.

2 Results

TABLE 7.1 Anti-inflammatory action of CMS-030 on carrageenin inducedfoot swelling in rats Inhibition Index Pre-challenge Swelling (cm) (%)Group Dose n circumference (cm) 0.5 hr 0.5 hr CMS-030   2 μg/kg/d 102.78 ± 0.06 3.20 ± 0.87** 37.0** CMS-030  10 μg/kg/d 10 2.76 ± 0.03 3.10± 0.10** 50.2** CMS-030  20 μg/kg/d 10 2.74 ± 0.04 3.12 ± 0.05** 43.8**DXM 0.2 mg/kg/d 9 2.49 ± 0.05 2.74 ± 0.10** 63.0** Saline 0.5 ml/d 102.80 ± 0.06 3.47 ± 0.08 — **compared with the saline control group, P <0.01.

Conclusion

Carrageenin induced foot swelling in rats was an established animalmodel of acute inflammation in vivo, and was used to evaluate theanti-inflammatory effects of drugs^([2]). Carrageenin can induceover-synthesis of prostaglandin in the inflammation site. Together withother vasoactive substances, the over-produced prostaglandin will inducelocal swelling. CMS-030 was found to be able to inhibit the footswelling induced by carrageenin in rats, with statistical significance(P<0.01). CMS-030 has therefore been shown to have anti-inflammatoryproperties.

References:

The following references are incorporated herein by reference in theirentireties.

-   [1] Li Jinhua, Zhang Huiqing, Zheng Kezhi, et al Inhibitory effects    of Orgotein on the swelling of hind paw in rats induced by    carrageenin. Suzhou University Journal of Medical Science, 2002,    Vol, 22(4):386-388.-   [2] Huang Zhili, Kagoshima Masatoyo, Kagawa Eiichiro,    Anti-inflammatory and ulcerogenic effects of 3-(N,N-diethylamino)    propylindometacin HCl. Acta Pharmacologica Sinica, 1997, Vol,    18(4):306-308.

Example 8 Effect of CMS-0300N Obesity

Objective:

To determine anti-obesity effect of CMS-030 using over-feed rat as theanimal model.

Methods:

An obesity model was developed by feeding rats with a high nutritiondiet for 6 weeks. The rats were then treated with CMS-030 (subcutaneous,at dosages of 150, 300, and 600 μg/kg/day), or with saline for 4 weeks.The rats were weighed once per week, and were killed for the measurementof abdominal and testicular fat pads and blood lipid at the end of theexperiment.

Results:

CMS-030 was found to be able to decrease the body weight, fat padindexes, serum triglyceride level, and total serum cholesterol level inrats, with statistical significance compared with the saline controlgroup (P<0.05).

Conclusion:

CMS-030 has anti-obesity properties, and may be used in the managementof diet induced obesity disorders.

Materials and Methods 1 Materials 1.1 Test Substance and Animal

CMS-030 was custom synthesized by Shenzhen Kangzhe Pharmaceutical Co.Ltd., Shenzhen, PR China.

Sprague-Dawley (SD) rats, specific pathogen free grade, weighing 135±15g, male: The Experimental Animal Center of First Military MedicalUniversity.

1.2 Reagent Kits

Triglyceride kit: Shanghai Rongcheng Biotechnology Laboratory.

Total serum cholesterol kit: Zhongsheng Beikong Biotechnology HoldingLtd.

2 Methods

The prescriptions of high and normal nutrition feed were prepared incompliance with The Guideline for Pre-clinical Research of Anti-obesityDrug, issued by State Food and Drug Administration, PR China(SFDA)^([1]).

The obesity model^([2]) was set up by feeding rats with a high nutritiondiet for 6 weeks. Rats served with normal diet were used as normalcontrol. The obese rats were then treated with CMS-030 (150, 300, and600 μg/kg/day, once daily, subcutaneous) or saline for 4 weeks. All ratswere on normal diet during test substance treatment. The rats wereweighed weekly and killed at the end of the experiment for themeasurement of abdominal and testicular fat pads, and the weights ofspleen, liver, kidney, and thymus. The rat blood was also drawn foranalysis of blood lipids.

Index of fat pad was calculated as: fat pad weight (g)/body weight(g)×1000.

Index of organ was calculated as: organ weight (g)/body weight (g)×1000.

3 Statistical Analysis

The data are presented as mean±standard deviation. A paired t test orsingle-factor ANOVA was applied in comparison within each group orbetween groups using the software DAS (Drug And Statistics Ver1.0).P<0.05 was taken as statistically significant.

4 Results

TABLE 8.1 Effect of CMS-030 on body weight of rats (unit: gram) Group n0 week 1 week 2 week 3 week 4 week Normal control 10 224.6 ± 15.6* 252.0± 18.0* 277.9 ± 20.2* 305.0 ± 26.9* 332.0 ± 22.7* Saline control 10342.6 ± 23.8 386.2 ± 26.9 386.1 ± 24.6 410.8 ± 25.7 408.8 ± 24.5CMS-030: 600 μg/kg/day 12 339.0 ± 27.0 365.4 ± 25.7 355.1 ± 24.6* 361.7± 32.6* 369.9 ± 30.0* CMS-030: 300 μg/kg/day 12 338.0 ± 22.8 369.7 ±26.1 359.6 ± 23.7* 364.3 ± 22.6* 366.3 ± 24.5* CMS-030: 150 μg/kg/day 12327.0 ± 26.9 348.9 ± 23.9* 346.5 ± 22.7* 351.8 ± 25.0* 354.0 ± 26.3*Compared with saline control: *p < 0.05

TABLE 8.2 Effect of CMS-030 on fat pad index of rat Index of Index ofGroup n body fat pad testicular fat pad Normal control 10 4.3 ± 1.2* 4.6± 0.9* Saline control 10 6.0 ± 1.8 5.5 ± 0.9 CMS-030: 600 μg/kg/day 124.5 ± 1.1* 5.0 ± 0.7* CMS-030: 300 μg/kg/day 12 3.8 ± 2.1* 4.0 ± 1.1*CMS-030: 150 μg/kg/day 12 3.9 ± 1.6* 3.6 ± 0.9* Compared with salinecontrol: * p < 0.05

TABLE 8.3 Effect of CMS-030 on blood triglyceride (TG) and totalcholesterol (TCH) of rats Group n TG (mg/dL) TCH (mg/dL) Saline control8 101.8 ± 31.2  333.3 ± 24.5 Normal control 8 36.4 ± 9.1* 225.7 ± 55.0*CMS-030: 600 μg/kg/day 12 40.6 ± 7.9* 228.1 ± 39.8* CMS-030: 300μg/kg/day 11 32.7 ± 6.7* 234.9 ± 39.1* CMS-030: 150 μg/kg/day 12  51.5 ±19.4* 270.9 ± 33.0* Compared with saline control: * p < 0.05

TABLE 8.4 Effect of CMS-030 on food intake by rats (unit: g/day) GroupWeek one Week two Week three Week four Normal 13.2 ± 1.3* 18.3 ± 6.1*18.6 ± 0.35 17.6 ± 2.7 control Saline 11.7 ± 2.3 15.5 ± 3.0 18.21 ±0.43  18.4 ± 0.59 control CMS-030: 13.8 ± 5.4 15.9 ± 1.7 18.2 ± 0.6918.5 ± 0.61 600 μg/kg/ day CMS-030: 13.2 ± 4.8 16.9 ± 1.6 18.1 ± 0.7418.4 ± 0.59 300 μg/kg/ day CMS-030: 13.2 ± 3.1 16.1 ± 1.8 18.3 ± 0.6717.8 ± 0.53* 150 μg/kg/ day Compared with saline control: * p < 0.05

TABLE 8.5 Effect of CMS-030 on organs indexes of rats Group n Thymusindex Liver index Spleen index Saline control 12 1.1 ± 0.3 25.5 ± 2.32.0 ± 0.3 Normal control 8 1.3 ± 0.3 25.5 ± 1.2 2.4 ± 0.2* CMS-030: 600μg/kg/ 12 1.1 ± 0.2 24.3 ± 2.2 2.0 ± 0.2 day CMS-030: 300 μg/kg/ 12 1.0± 0.3 23.1 ± 1.3* 2.0 ± 0.2 day CMS-030: 150 μg/kg/ 12 0.9 ± 0.2 25.9 ±3.6 2.0 ± 0.2 day Compared with saline control: * p < 0.05

Conclusion

CMS-030, at a suitable dosage, was found to be able to induce weightloss and reduce blood lipid levels in a nutritionally obese rat, withstatistical significance compared with saline control (P<0.05). Appetitewas not significantly affected during the administration of thesubstance. CMS-030 may be useful in the management of nutrition relatedobesity and hyperlipidemia.

References:

The following references are incorporated herein by reference in theirentireties.

-   1. SFDA, PR China. The guideline for pre-clinical research of new    drugs. 1993.193-194.-   2. Bays HE. Current and investigational anti-obesity agents and    obesity therapeutic treatment targets. Obes Res. 2004; 12    (8):1197-1211.

Example 9 Effects of CMS-0300N Delayed Hypersensitivity in Mice

Objective:

To investigate the inhibitory effect of CMS-030 on delayedhypersensitivity (DTH) in mice.

Methods:

2,4-dinitrofluorobenzene (DNFB) induced ear swelling was used todemonstrate the immunosuppressive effects of CMS-030.

Results:

At 10 μg/kg/day, CMS-030 was found to be able to suppress DNFB-inducedear swelling in mice, with statistical significance compared with thesaline control (P<0.01).

Conclusion:

CMS-030 has immunosuppressive properties and may be useful for themanagement of immunity related disorders.

1 Materials and Methods 1.1 Drugs and Reagents

CMS-030: Custom synthesized by Shenzhen Kangzhe Pharmaceutical Co. Ltd.,Shenzhen, PR China.

2,4-dinitrofluorobenzene (DNFB): Smack Co. Ltd.

Sodium sulfide (Na₂S): Tianjin Beilian Chemical Co. Ltd., Tianjin, PRChina.

1.2 Animals

Balb/c mice, specific pathogen free (SPF) grade, 6-8 weeks old, weighing18-22 g: Military Medical Academy of Science, PR China.

1.3 Methods

BALB/c mice were randomized into groups of saline control (0.5 ml/day)and CMS-030 (10 μg/kg/day). The test substance was dissolved in 0.5 mlsaline, and was administrated intraperitoneally once per day for twoweeks before sensitization.

The mice were depilated with 8% Na₂S solution at the abdomen one daybefore the sensitization. DNFB was dissolved in acetone/olive oil (4:1)to final concentration of 1% and 50 μl was applied to the depilated areafor sensitization. Four days after the initial sensitization, 10 μl ofthe 1% DNFB solution was applied topically to the right ear to elicitdelayed hypersensitivity inflammation. The same volume of solventwithout DNFB was applied to the left ear as the baseline. 24 hrs later,a 6 mm diameter punch was used to collect a piece of ear tissue from thesame location of the left and right ear, and the tissue was weighedaccurately. The ear swelling was calculated by subtracting the weight oftissue from the right ear with that of the left ear from the samemouse^([1]). Inhibit rate (%)=(Ear swelling of saline control−Earswelling of test group)/Ear swelling of saline control×100%.

1.4 Statistical Analysis

Statistical analysis was performed with One-Way ANOVA by SPSS.

2 Results

TABLE 9.1 The inhibitory effects of CMS-030 on DH in mice Group Dosage nEar swelling (mg) Inhibit rate (%) CMS-030  10 μg/kg/day 17 3.69 ± 2.31*46.5* Saline 0.5 ml/day 18 6.91 ± 2.50 — *Compared with saline controlgroup, p < 0.01

3 Conclusion

CMS-030 was found to be able to inhibit the delayed hypersensitivityresponse of mice to DNFB, with statistical significance compared withthe saline control (P<0.05). This showed that CMS-030 may be used forthe management of hypersensitivity related immunity disorders.

Reference:

The following reference is incorporated herein by reference in itsentirety.

-   [1] Li Weidong, Ren LianSheng, Lin Zhibin, et al. Preliminary study    on immunomodulating actions of Actarit in mice. Journal of Beijing    Medical University, 2000, Vol, 1(32): 1-3.

Example 10 Studies on the Immuno-Suppressive Properties of CMS-030 InVivo

Objective:

To investigate the anti-allograft-rejection properties of CMS-030 andthe possible mechanisms of action.

Methods:

The immuno-suppressive properties of CMS-030 was observed with theT-lymphocyte proliferation test and Mixed lymphocyte reaction (MLR) invitro. The effect of CMS-030 on the survival of allograft was observedon mice skin and cardiac muscle allograft animal model in vivo. Theeffect of CMS-030 on the transformed T-cell and spleen cell IL-2secretion of the allograft recipient was also observed.

Conclusion:

CMS-030 was observed to have anti-allograft-rejection properties. Thismay be mediated via suppression of T-lymphocyte activities and IL-2secretion by lymphocytes.

1 Materials and Methods 1.1 Animals

Five week old specific pathogen-free female and male Balb/c (H-2^(b))and C₅₇BL/6J (H-2^(d)) mice: The Institute for Laboratory Animals ofMilitary Medical Academy of Science (Beijing, China). Half male and halffemale.

C57BL/6 new born mice were from self-breeding.

1.2 Drugs and Other Reagents

CMS-030: Custom synthesized by Shenzhen Kangzhe Pharmaceutical Co. Ltd.,Shenzhen, PR China.

Cyclosporine (CsA): Novartis Pharmaceutical Co. Ltd., Basel,Switzerland. Dissolve to 0.5 ml final volume for all CsA groups.

Bovine serum, RPMI-1640, Hank's solution: GIBCO®, Invitrogen, Carlsbad,Calif., USA.

MTT, ConA: Sigma Chemical Co., St. Louis, Mo., USA.

NaS: Tianjin Beilian Fine Chemicals Co., Ltd., Tianjin, PR China.

Mouse IL-2 Elisa kit: R&D Systems Inc., Minneapolis, Minn., USA.

1.3 Grouping of Animals

For the in vivo tests, the mice were randomized into groups of

CMS-030 (10 μg/kg/day).CMS-030 (2 μg/kg/day).CsA (10 mg/kg/day).Normal saline (0.5 ml/day).

The solutions were applied by intraperitoneal injection, once per dayfor 5 days before transplantation and continued for another 20 daysafter the surgery.

1.4 Effect of CMS-030 on T-Lymphocyte Proliferation In Vitro^([1])

Healthy Balb/c mice spleens were dissected aseptically and immersed inice-cold D-Hank's solution. Spleen cells were prepared by disrupting thespleen with frosted glass slides in RPMI-1640. The cells were washedtwice in RPMI-1640 at 4° C. and 1,200 rpm for 10 min. The cells werethen counted and adjusted to the concentration required for each assay.Cell viability in the experiment was determined by trypan blue exclusionand should be greater than 95%.

The spleen cells from healthy mice were cultured in 96-well plates,adjusted to a concentration of 4×10⁵ per well. CMS-030 was added to thewells of final concentration of 40 μg/ml, 8 μg/ml, 1.6 μg/ml, and 0.32μg/ml. The plates were incubated in a humidified atmosphere at 37° C.,5% CO₂ for 68 hr in the presence of concanavalin A (ConA) at a finalconcentration of 5 μg/ml. In the positive control group, no CMS-030 wasadded. In the negative control group, neither ConA nor CMS-030 wasadded. The proliferation of lymphocytes was measured by the3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)colorimetric assay at the end of incubation. 20 μl of MTT was added toeach well, and the plates were further incubated for 4 hr. Then, 100 μlof 0.04 M HCl in isopropyl alcohol was added to each well in order tosolubilize the formazan precipitate. The OD of each sample was measuredspectrophotometrically at 570 nm referenced at 630 nm

1.5 Effect of CMS-030 on Mixed Lymphocyte Reaction (MLR) In Vitro^([2])

Healthy Balb/c mice spleen cells were washed with ice-cold D-Hank'ssolution and resuspended in RPMI-1640 to 8×10⁶/well to form theresponder cells. Spleen cells suspension from healthy C₅₇BL/6J mice wereincubated with mitomycin (25 μg/ml) for 45 minutes in RPMI 1640 at 37°C. and in 5% CO₂, then washed with RPMI-1640 and resuspended to8×10⁶/well to form the stimulant cells. Using a 96 wells culture plate,to the test groups, add in 100 μg responder cells and 100 μg stimulantcells, and 20 μl CMS-030 to final concentrations of 40 μg/ml, 8 μg/ml,1.6 μg/ml, or 0.32 μg/ml. To the positive control group, 100 μl ofresponder cells, 100 μl stimulant cells, and 20 μl RPMI-1640 were added.To the negative control group, 200 μl stimulant cells and 20 μlRPMI-1640 were added. Six parallel wells were used for each combination.After 5 days of incubation at 37° C. and 5% CO₂, the proliferation oflymphocytes was measured by the MTT colorimetric assay.

1.6 The Effect of CMS-030 on the Survival of Heart Transplant InVivo^([3])

The donor hearts were dissected from 24 hour old newborn C₅₇BL/6J mice.The hearts were immersed in D-Hank's solution and the blood from thecardiac cavity was cleared. The hearts were transplanted subcutaneouslyin the ear pinnae passages of healthy adult Balb/c recipients. The airin the heart and the passage was expelled by slight pressure. Startingfrom the sixth day after transplantation, the electrocardiogram (ECG) ofthe transplanted heart was taken daily. Absence of ECG signal for 3consecutive days showed that the transplantation surgery wasunsuccessful and the mice were excluded from the statistics analysis.The ECG was followed daily and time to rejection was taken as the day onwhich the ECG signal disappeared. Test substances were administeredintraperitoneally starting from 6 days before surgery. There were 10mice per group. Cyclosporine A was administered at 10 mg/kg/day, CMS-030at 10 μg/kg/day or 2 μg/kg/day, and saline at 0.5 ml/kg/day. Statisticalanalyses were performed using Kaplan Meier log-rank test comparisonswith saline-treated group.

1.7 Effect of CMS-030 on the Survival of Skin Allograft In Vivo^([4])

A patch of hair at the back of Balb/c mice was removed by 8% Na₂Ssolution. On the next day, a wound bed of approximately 1 cm² wasproduced by removing the skin surgically, and then a piece offull-thickness tail skin of 1 cm² from sex-matched donor C₅₇BL/6J micewas placed onto the wound bed. The surgical site was covered andprotected with a layer of paraffin gauze, and a covering plaster wasapplied. The plaster was removed 8 days after transplantation, and therecipient mice were monitored daily for the viability of the allograft.The end point of rejection was taken as only less than 10% of theallograft remained viable. Intraperitoneal treatment with testsubstances was started 6 days before surgery. There were 10 mice pergroup. Cyclosporine A was administered at 10 mg/kg/day, CMS-030 at 10μg/kg/day or 2 μg/kg/day, and saline at 0.5 ml/kg/day. The treatment wascontinued for another 20 days after surgery.

1.8 Effect of CMS-030 on T Lymphocyte Proliferation In Vivo^([5])

Spleen cells from the skin graft recipient mice were isolated andre-suspended in RPMI-1640 to 4×10⁶/ml. 100 μl/well cells were added intoa 96-well plate. To the test wells, 100 μl ConA was added to the finalconcentration of 5 μg/ml. To the control wells, 100 μl RPMI-1640 wasadded instead. Four parallel wells per condition. The cells wereincubated for 68 hrs at 37° C. and 5% CO₂. The proliferation oflymphocytes was measured by MTT colorimetric assay. The OD of eachsample was measured spectrophotometrically at 570 nm referenced at 630nm. The data were expressed as the stimulation index, which was the ODof the test group divided by the OD of the control group.

1.9 Effect of CMS-030 on IL-2 Levels In Vivo^([6])

Spleen cells from the skin graft recipient mice were isolated andre-suspended in RPMI-1640 to 2×10⁶/ml. 1.5 ml/well cells were added intoa 24 wells plate and incubated for 24 hrs for attachment. 100 μl ConAwas added to the final concentration of 10 μg/ml. The cells wereincubated for another 48 hrs and the supernatant subsequently collectedafter centrifugation. IL-2 levels in culture supernatants weredetermined by ELISA.

1.10 Statistical Analysis

A Kaplan Meier log-rank test comparison with control group was used forallograft survival time analysis. Comparison of means with variances by2-tailed Student t test was used for other experiments.

2 Results

TABLE 10.1 Effect of CMS-030 on T-lymphocyte proliferation in vitroGroup Dose of drugs No. OD CMS-030   40 μg/ml 6 0.332 ± 0.062* CMS-030  8 μg/ml 6 0.281 ± 0.041* CMS-030  1.6 μg/ml 6 0.311 ± 0.027* CMS-0300.32 μg/ml 6 0.315 ± 0.043* Positive control — 6 0.421 ± 0.055 Negativecontrol — 6 0.109 ± 0.003* *Compared with saline control group, p < 0.01

TABLE 10.2 Effect of CMS-030 on Mixed lymphocyte reaction (MLR) in vitroGroup Dose of drugs No. OD CMS-030  40 μg/ml 6 0.192 ± 0.019* CMS-030  8 μg/ml 6 0.285 ± 0.004* CMS-030 1.6 μg/ml 6 0.361 ± 0.036* Positivecontrol — 6 0.440 ± 0.043 Negative control — 6 0.145 ± 0.019 *Comparedwith saline control group, p < 0.01

TABLE 10.3 The effect of CMS-030 on the survival of heart transplant invivo Group Dose of drugs No. Mean survival time (day) CMS-030  10μg/kg/d 9 12.0 ± 2.2* CMS-030   2 μg/kg/d 9 11.5 ± 1.9* CsA  10 mg/kg/d8 13.8 ± 1.3* Saline 0.5 ml/d 10  9.1 ± 1.4 *Compared with salinecontrol group, p < 0.05

TABLE 10.4 Effect of CMS-030 on the survival of skin allograft in vivoGroup Dose of drugs No. Mean survival time (day) CMS-030  10 μg/kg/d 914.1 ± 1.2* CMS-030   2 μg/kg/d 10 13.4 ± 1.5* CsA  10 mg/kg/d 9 15.0 ±1.4* Saline 0.5 ml/d 9 11.0 ± 1.3 *Compared with saline control group, p< 0.01

TABLE 10.5 Effect of CMS-030 on T lymphocyte proliferation in vivo GroupDose of drugs No. Stimulation index CMS-030  10 μg/kg/d 9 1.6 ± 0.3*CMS-030   2 μg/kg/d 10 1.9 ± 0.5* CsA  10 mg/kg/d 9 1.8 ± 0.3* Saline0.5 ml/d 9 2.3 ± 0.5 *Compared with saline control group, p < 0.01

TABLE 10.6 Effect of CMS-030 on IL-2 levels in vivo Group Dose of drugsNo. IL-2 (pg/ml) CMS-030  10 μg/kg/d 9 599.0 ± 121.8* CMS-030   2μg/kg/d 10 577.4 ± 163.1* CsA  10 mg/kg/d 9 595.2 ± 162.8* Saline 0.5ml/d 9 787.4 ± 227.8 *Compared with saline control group, p < 0.01

Conclusion

At the concentration of 0.32 μg/ml to 40 μg/ml, CMS-030 was found to beable to statistically significantly inhibit ConA induced T lymphocytesproliferation in vitro, showing that CMS-030 can inhibit theproliferation of T lymphocytes in vitro. MLR is an experiment in vitroto determine the response of lymphocytes to different HLA-II molecules,and is a model for the prediction of rejection potential after organtransplantation^([7]). At the concentration of 1.6 μg/ml to 40 μg/ml,CMS-030 was found to be able to statistically significantly inhibitmixed lymphocyte reaction, indicating that CMS-030 can decrease therejection potential after organ transplantation.

The heart transplant and skin allograft experiments were animal modelsfor the study of the suppression of rejection after transplant^([7]).CMS-030 at dosage of 2 μg/kg/day and 10 μg/kg/day was found to be ableto prolong the survival of the transplant, with statisticalsignificance, showing that CMS-030 can suppress rejection of thetransplant by the host immune response. Analysis of the spleen cellsisolated from the skin graft recipient mice showed that CMS-030 was ableto suppress the activation of the lymphocyte and the secretion of IL-2by the T lymphocytes, both with statistical significance, showing thatthe prolongation of survival of the allograft was achieved bysuppression of the immune response of the recipient animal.

References:

The following references are incorporated herein by reference in theirentireties.

-   [1] Roma Kalra, Shashi P. Singh, Juan C, et al Immunosuppressive and    Anti-Inflammatory Effects of Nicotine Administered by Patch in an    Animal Model. Clinical and Diagnostic Laborarory Immunology, May    2004, 563-568.-   [2] Dubey D P, Yunis I, Yunis E J, et al. Cellar typing: mixed    lymphocyte response and cell mediated lympholysis. American Society    for Microbiology, 1986, 847-848.-   [3] Vakeva A Laurila P, Meri S, et al. Regulation of complement    membrane attack complex formation in myocardial infarction. Am J    Pathol, 1993, 143:65.-   [4] Ming Jiankuo, Wang Xingbing, Huang Baojun, et al. Peptide    Nucleic Acid Antisense Prolongs Skin Allograft Survival by Means of    Blockade of CXCR3 Expression Directing T Cells into Graft. The    Journal of Immunology, 2003, 170:1556-65.-   [5] María A. Puertollano, Manuel A. de Pablo, et al. Relevance of    Dietary Lipids as Modulators of Immune Functions in Cells Infected    with Listeria monocytogenes. Clinical and Diagnostic Laboratory    Immunology. 2002, 9:352-357.-   [6] Mayumi H, Himeno K, Shin T, et al. Drug-induced tolerance to    allografts in mice. Immunobiology, 1985, 169(2):147-161.-   [7] Rene J. Duquesnoy Li Y P. Transplantation immunobiology. 2002,    10:5-7.

Example 11 The Effect of Peptides on Exercise-Induced Fatigue of Mice

Objective:

To investigate the anti-fatigue effects of peptides on Balb/c mice.

Methods:

Male Balb/c mice swimming time was used as the animal model for studyingthe anti-fatigue effect of peptides.

Results:

CMS-001.30 and CMS-001.31 was found to be able to lengthen the swimmingtime of mice, with statistical significance compared with the control(P<0.01).

Conclusion:

CMS001-30 and CMS001-31 has anti-fatigue properties and may be used onthe management of fatigue related disorders.

1 Material and Methods 1.1 Drug and Reagents

CMS-001.30 and CMS-001.31 were custom synthesized by Shenzhen KangzhePharmaceutical Co. Ltd., Shenzhen, PR China.

Erythropoietin (EPO): Japan Kunpeng Medical Corporation.

1.2 Animals

Balb/c mice, male, specific pathogen free (SPF) grade, weighing 18-22 g:Academy of Military Medicines and Sciences Experimental Animal Center,PR China.

1.3 Groupings and Method^([1])

Balb/c male mice were randomized into groups of CMS-001.30 (20μg/kg/day), CMS-001.31 (20 μg/kg/day), EPO (1000 u/kg/day, three timesper week), and saline control. The test substance was dissolved in 0.5ml saline and applied intraperitoneally once per day for 30 continuousdays (EPO was replaced with saline if EPO was not applied). On the tenthday, the mice were trained to swim for 10 min at water temperature of25±1° C. 30 min after the last test substance administration, the micewere placed to swim in the swimming tank (50 cm×50 cm×40 cm). The depthof water was 30 cm and water temperature 25±1° C. The limbs of the micewere kept moving in the whole process. The swimming (min) time of themice until death was recorded.

Exhaustive swimming time extension rate (ESTR) (%)=(Mean exhaustiveswimming time of test group−Mean exhaustive swimming time of salinecontrol)/(Mean exhaustive swimming time of saline control)×100%.

1.4 Statisitics

Difference between groups were analyzed by ANOVA Analysis of variance

2 Results

TABLE 11.1 The effect of peptides on the exhaustive swimming time ofmice Swimming time Groups Dosages No. (min) ESTR (%) CMS-001.30  20μg/kg/day 20 186.4 ± 15.2* 88.3* CMS-001.31  20 μg/kg/day 20 174.3 ±29.2* 76.0* EPO 1000 u/kg/day, 20 126.1 ± 20.4* 31.3* 3 times per weekSaline   0.5 ml/day 20 99.0 ± 11.2 — *compared with the saline group P <0.01

3 Conclusion

CMS001-30 and CMS001-31 were found to have anti-fatigue properties andcan be used for the management of fatigue related disorders.

Reference:

The following reference is incorporated herein by reference in itsentirety.

-   1. Mizunoya W, Oyaizu S, Ishihara K, et al. Protocol for measuring    the endurance capacity of mice in an adjustable-current swimming    pool. Biosci Biotechnol Biochem. 2002 May; 66(5):1133-1136.

Based on the above information, various pharmaceutical formulation canbe made from the disclosed peptides. The pharmaceutical formulation mayinclude any of the known pharmaceutical carriers. Examples of suitablecarriers include any of the standard pharmaceutically accepted carrierknown to those skilled in the art. These include but are not limited to,physiological saline solution, water, emulsions including oil and watermixtures or triglyceride emulsions, and other types of agents, fillers,coated tablets and capsules. The appropriate carrier may be selectedbased on the mode of administration of the pharmaceutical composition.

The pharmaceutical formulation can be administered via intravenousinjection, intramuscular injection, intraperitoneal injection,subcutaneous injection, and subcutaneous implantation. The peptide mayalso be administered in any form of oral administration such as, forexample, a tablet, capsule, suspension, solution, and the like, in theusual form without modification or in slow release form, or with orwithout gastro-enteric protection. The peptide can also be applied inany form of topic application such as an ointment, cream, gel, etc.,with or without a transdermal facilitating device. The peptide may alsobe interpreted into its genetic sequence and cloned into an expressionsystem, on its own or in combination with other peptide sequences, togenerate a resulting peptide molecule to make use of the activity of thepeptide as described herein.

The dose of each peptide may be 1 ng-10 g per kg body weight. Apreferred dose is 10 ng-10 mg per kg, and more preferably 1 μg-1 mg perkg for an injection mode of administration. However, the effective dosecan be as low as 1 ng per kg body weight, since one or more of thepeptides may operate through receptors that will induce a cascade ofnormal physiological responses. Alternatively, one or more of thepeptides can just be an initiator for a whole cascade of reaction. Foran oral intake, the amount may be 1 ng-10 g per day per kg body weight,more preferably 0.1 μg-1 g per day per kg body weight and even morepreferably 1 μg-10 mg per day.

Gene therapy based on the above peptide sequences may be performed basedon methods known in the art, and also based on patent publication WO03/006492A2, which is incorporated by reference in its entirety herein.The peptides may also be conjugated to other enhancer molecules based onthe teaching disclosed in patent publication WO2004/055042A1 and whichis incorporated by reference in its entirety herein.

References:

The following references are incorporated herein by reference in theirentireties.

-   1. Principles of Pre-clinical Research of New Drugs, People's    Republic of China. 1993, 7:134-135.-   2. Shuyun Xu, Rulian Bian, Xiu Chen. Methodology of pharmacological    experiment. People's Health Publishing House. 1991, 1221-1234.-   3. Principle of new drug research in pre-clinic issued by Ministry    of Health, People's Republic of China. 1993, 7:140.-   4. Jinsheng He, Ruizhu Li, Tingyi Zong. The study on MTT reduction    method of testing NK cell activity. China Immunology Journal. 1996,    1(6):356-358.-   5. Qian Wang. Modern medical experiment method. People's Health    Publishing House. 1998, 482-483.-   6. Principle of new drug research in pre-clinic issued by Ministry    of Health, People's Republic of China. 1993, 7:141.-   7. Principle of new drug research in pre-clinic issued by Ministry    of Health, People's Republic of China. 1993, 7:132-133.-   8. Principle of new drug research in pre-clinic issued by Ministry    of Health, People's Republic of China. 1993, 7:128-129.-   9. Yuanpei Zhang, Huaide Su. Phamalogical experiment (second    edition). People's Health Publishing House. 1998, 137-138.-   10. Jiatai Li, clinical pharmacology (second edition). People's    Health Publishing House. 1998, 1338-1339.

Example 12 Delivery of Peptides Through Genetically EngineeredLactobacillus Bacterial Species

The following is provided as one exemplary method to deliver peptides ofthis invention to a host as described above. A DNA sequence that encodesone of the peptides listed in table A above is synthesized by chemicalmeans and this DNA sequence is inserted into an expression vector usingstandard techniques of genetic engineering familiar to those skilled inthe art. The expression vector selected contains a constitutive promoterfunctional in Lactobacilli, a multiple cloning site for the introductionof DNA sequences in a specific 5′ to 3′ orientation as well as aselectable marker gene that confers resistance to an antibiotic (to aidin cloning procedures) and may comprise other sequences to assist in theproduction and/or secretion of the peptides, such as signal peptidesequences. An example of such a vector is provided by U.S. Pat. No.5,592,908, to Pavla, which is hereby incorporated by reference in itsentirety. Briefly, this patent discusses several known promoters thatfunction in Lactobacillus species, as well as a method for discoveringnovel promoters in said bacteria, any of which may be operably linked toa nucleic acid encoding a peptide of the present invention to expressthe peptide in Lactobacilli. A nucleic acid encoding a signal peptide,such as peptides comprising of 16 to 35 mostly hydrophobic amino acidsthat are active in Lactobacillus lactis described in U.S. Pat. No.5,529,908, cited above, is interposed between the promoter and thenucleic acid encoding the peptide of the present invention such that thenucleic acid encoding the signal peptide is in frame with the nucleicacid encoding the peptide of the present invention.

In addition to the coding sequence of the peptide, the DNA sequencesynthesized may comprise sequences to aid in the ligation and cloning ofsaid DNA into the expression vector. For example, restriction enzymerecognition sites that correspond to ones found in the multiple cloningsite of the vector can be incorporated into the synthesized DNA at the5′ and 3′ ends of the sequence, so that the sequence can be cloned inproper orientation within the vector. Both the vector and thesynthesized DNA are digested with the particular restriction enzymes,then purified. Ligation reactions with the vector and the synthesizedDNA are followed by transformation into a suitable strain of E. Coli.The transformed bacteria are plated on media containing the antibioticto which the vector confers resistance. A colony of transformed bacteriais selected for growth cultures and plasmid preparation procedures; thepresence of the synthesized DNA in the correct orientation is confirmed.

This expression vector is then transformed into a bacterial host cell ofa Lactobacillus species, such as L. acidophilus. Transformed cells areselected for by virtue of the selectable marker found within the vectorsequence and the secretion of the peptide may be verified by performinga western blot, performing gel electrophoresis of peptides present inthe growth medium or other standard techniques. A transformed colony ofbacteria is chosen and used to prepare large-scale cultures of thegenetically engineered bacteria. A culture of the genetically engineeredbacteria expressing the desired peptide is grown up and at least aportion thereof is administered to the alimentary canal, vagina, tracheaor other area of the host organism in which the bacteria are able toreplicate. If desired, the bacterial cultures can be treated in avariety of ways to produce a supplement for enteric consumption by thehost. These treatments include lyophilization or other methods ofpreserving the bacteria, in addition to combining the bacteria withcarrier agents, such as solutions, solvents, dispersion media, delayagents, emulsions and the like. The use of these agents to preparesupplements is well known in the art. For example, the bacteria can beused to make cultured milk products or other foodstuffs for humanconsumption, such that the organism expressing the peptide colonizes thegut of the host organism. A number of different methods forincorporating specific strains of lactic acid bacteria into foodstuffssuch as yogurt, kimchee, cheese and butter are disclosed in U.S. Pat.No. 6,036,952, to Oh, which is hereby incorporated by reference in itsentirety. Upon consuming the bacteria through one of any number ofroutes, the engineered organisms can colonize the gut and allow thepresentation and/or absorption of the peptides of this invention via themucosal layer of the gut.

Example 13 Delivery of Peptides Through a Genetically Engineered Form ofBacillus subtilis

The following is provided as another exemplary method to deliverpeptides of this invention to a host as described above. A DNA sequencethat encodes one of the peptides listed in table A above is synthesizedby chemical means and this DNA sequence is inserted into an expressionvector via techniques of genetic engineering, all techniques being knownin the art. The expression vector selected comprises a shuttle vector,such as pTZ18R (Pharmacia, Piscataway, N.J.), capable of beingpropagated in both E. Coli and B. Subtilis and containing an antibioticresistance gene for selecting colonies of transformed bacteria. Thisvector can contain a constitutive promoter active in B. subtilis, suchas a promoter derived from the Sac B gene of B. subtilis as well as anucleotide sequence encoding a signal peptide active in B. subtilis thatdirects efficient export of expressed heterologous proteins from thebacterial cell. An example of such a vector is disclosed in U.S. Pat.No. 6,268,169, to Fahnestock, the disclosure of which is incorporatedherein by reference in its entirety. Briefly, as detailed above, the DNAencoding a peptide of this invention will be synthesized withrestriction enzymes sites and/or other sequences to facilitate cloningof the DNA through techniques familiar to those with skill in the art.After transformation into E. Coli., plating, selection and propagationof the plasmid to create a plasmid stock, the plasmid is then betransformed into B. subtilis and transformants are selected by virtue ofresistance to an antibiotic in the plating media.

Peptide production in and secretion from the genetically engineered B.subtilis is verified using techniques well known to those with skill inthe art, such as radiolabeling of peptides for autoradiographicdetection after SDS-PAGE anaylsis or Western blotting.

A culture of genetically engineered bacteria is grown up and at least aportion thereof is administered to the alimentary canal, vagina, tracheaor other area of the host organism in which the bacteria are able toreplicate.

Example 14 Delivery of Peptides Through Genetically EngineeredSaccharomyces Yeast Species

The following is provided as another exemplary method to deliverpeptides of this invention to a host as described above. A DNA sequencethat encodes one of the peptides listed in table A above is synthesizedby chemical means and this DNA sequence is inserted into an expressionvector via techniques of genetic engineering, all techniques being knownin the art. The expression vector selected comprises a stably maintainedyeast protein expression vector, comprising a constitutive yeastpromoter such as pADH1, sites for replication of the vector in bothyeast and E. Coli, a gene or genes that confer prototrophy to anauxotrophic yeast mutant for selection purposes, a multiple cloning site(MCS) and, if desired, sequences that code for a signal peptide. Vectorssuch as this are commercially available and well known in the art or canbe readily constructed using standard techniques After insertion of thesynthesized DNA into the yeast vector, transformation into E. Coli,plating of transformed E. Coli onto selective media, selection of atransformed bacterial colony and preparation of plasmid DNA from agrowth culture of bacteria from said colony, the vector is transformedinto Saccharomyces cerevisiae via well-known techniques such as lithiumacetate transformation or electroporation. The strain of Saccharomycescerevisiae selected for transformation is a mutant auxotrophic strainthat will require a gene on the plasmid in order to grow on minimalmedia plates. Transformed yeast colonies are isolated by plating theyeast on growth media lacking the gene provided on the vector. Onlythose yeast that have received the vector and its selective gene and areexpressing that gene product will be able to grow into colonies on theminimal media. Verification of peptide secretion can be obtained byperforming a Western blot, performing gel electrophoresis of peptidespresent in the growth medium or other standard techniques.

A transformed colony of yeast is chosen and used to prepare large scalecultures. A culture of the genetically engineered yeast expressing thedesired peptide is grown up and at least a portion thereof isadministered to the alimentary canal, vagina, trachea or other area ofthe host organism in which the bacteria are able to replicate. Ifdesired, the yeast cultures can be treated in a variety of ways toproduce a supplement for enteric consumption by the host. Thesetreatments include lyophilization or other methods of preserving yeast,in addition to combining the bacteria with carrier agents, such assolutions, solvents, dispersion media, delay agents, emulsions and thelike. The use of these agents to prepare supplements is well known inthe art. In another embodiment, the transformed yeast are used in thecreation of food products, such as fermented milk products like yogurtand kefir, by techniques known to those skilled in the art. As with livelactic acid bacterial cultures in these foodstuffs, the transformedyeast colonize the gut at least transiently and serve to presentpeptides to the host via the gut lumen.

While the present invention has been described using the aforementionedmethods and data and the specific example of the peptides describedherein in many cases, it is understood that this is an example only andshould not be taken as limitation to the present invention. It shouldalso be understood that these peptides described herein represent someembodiment of the present invention and the same principle of thepresent invention can also apply to other functionally equivalentpeptides that have been modified without affecting the biologicalfunction of these peptides. Furthermore, although the disease ordisorder described above for the medical application of these peptidesare recited to support their usefulness, these medical applications areused as non-limiting examples only and should not be used to limit thescope of the claims. It is clear that there are other possible/intendeduse of these peptides and their functional derivatives, such as but notlimited for use as a health food supplement to enhance or boost theimmune system, alleviate fatigue, reduce blood lactic acid of a normalperson or a patient with any infections. Any such uses also fall withinthe scope of the present invention.

As for the peptides with sequences that have been previously published,the present invention has provided new and unexpected uses therefor, andit is believed that these new indication support the use of some ofthese know peptides for industrial applications that were previouslyunforeseen. Beside new medical uses as described above and in theclaims, they can also be used as dietary or nutritional supplements forimproving conditions of a normal individual based on the teachingprovided herein.

What is claimed is:
 1. A method of modulating blood lactic acid level ina patient in need thereof comprising administering a pharmaceuticallyeffective dose of a biologically active peptide, said biologicallyactive peptide having an amino acid sequence comprising SEQ ID NO:
 1. 2.The method of claim 2 wherein said biologically active peptide having anamino acid sequence consisting of SEQ ID NO: 1.